Induction of mucosal tolerance to antigens

ABSTRACT

Induction of tolerance to antigens by mucosal, preferably oral, delivery of the antigen in combination with an immunomodulating compound producing micro-organism is disclosed. More specifically, Foxp3 +  and/or IL-10 and/or TGF-β producing regulatory T-cells are induced which are capable of suppressing undesired immune responses toward an antigen. The antigen is preferably delivered orally in combination with an immunosuppressing cytokine secreting micro-organism.

The present invention relates to the induction of tolerance to antigens,by mucosal, preferably oral delivery of the antigen in combination withan immuno-modulating compound producing micro-organism. Morespecifically, the invention relates to the induction of Foxp3⁺ and/orIL-10 and/or TGF-β producing antigen-specific regulatory T-cells,capable of suppressing undesired immune responses toward an antigen, byoral delivery of said antigen in combination with an immuno-suppressingcytokine secreting micro-organism.

FIELD OF THE INVENTION

The immune system has the task of distinguishing between self andnon-self. The mucosal immune system, present along the respiratory,gastrointestinal and genitourinary tracts, has the additional burden ofcoexisting with an abundance of bacteria and innocuous antigens, such asfood, airborne antigens or the commensally bacterial flora. A keyfeature of the mucosal immune system is its ability to remain tolerantto these antigens while retaining the capacity to repel pathogenseffectively. Introduction of antigen systemically, whether by injectionor injury, leads to local infiltration of inflammatory cells andspecific immunoglobulin production. By contrast, antigens introduced atmucosal surfaces, such as the gastrointestinal and genitourinary tracts,elicit active inhibition of the immune response to those antigenssystemically. The specific induction of these regulated responses byadministration of antigen through the gastrointestinal tract is known asoral tolerance. Oral administration of antigen can lead to systemicunresponsiveness and is an attractive alternative to immunosuppressivemedical inventions that have undesirable side-effects (such assteroids). The invention lies in particular in the field of low-dosetolerance, obtained by repeated exposure to low doses of antigen.Tolerance inductions via the mucosa have been proposed as a treatmentstrategy against autoimmune, allergic and inflammatory diseases.

STATE OF THE ART

Although oral tolerance was first described in 1911, it was not untilthe later 1970s that investigators started to address the mechanismsinvolved (Mayer and Shao, 2004a). Several mechanisms have been proposedfor the development of oral tolerance, ranging from the deletion ofanti-specific T-cells, over immune deviation and induction of anergy tosuppression by Tregs (Mucida et al., 2005). Most investigators agreethat there are two distinct ways of obtaining oral tolerance, thehigh-dose tolerance, obtained after a single high dose of antigen, whichis based on anergy and/or deletion (Friedman and Weiner, 1994), and thelow-dose tolerance, obtained by repeated exposure to low doses ofantigen, mediated by active suppression of immune responses by CD4⁺T-cells, including Foxp3⁺, IL-10 and/or TGF-β producing regulatoryT-cells. Importantly, regulatory T cells induced through mucosaltolerance have been shown to mediate bystander suppression, a processthrough which regulatory cells specific for one protein suppress theresponse of nearby effector cells to another protein. Bystandersuppression is an important feature of antigen-induced suppressionbecause the pool of antigens that induce organ-specific autoimmunity arelargely unknown, and it overrides the phenomenon of epitope spreading.Epitope spreading is a complication of autoimmune and allergic diseaseswhereby the initiating immune response expands with time to includeresponses to other antigens.

The role of dendritic cells in the induction of oral tolerance has beenalluded to through studies showing enhanced oral tolerance followingFlt3L-driven expansion of DC (Viney et al. 1998) and RANK-L-mediated DCactivation (Williamson et al., 2002) in vivo. In particular, immaturedendritic cells can mediate tolerance, presumably by induction ofregulatory T cells. Moreover, IL-10 can modulate the function ofimmature dendritic cells and inhibit their terminal differentiation,amplifying the local presence of tolerizing dendritic cells involved inthe induction of regulatory T cells (De Smedt et al. 1997).

Mucosal tolerance induction has been evaluated in numerous experimentalmodels of allergy and autoimmune disease, but clinical data from trialsin humans have been generally disappointing. A number of attempts havebeen made to deliver antigens, whether or not in combination with animmuno-modulating compound, in order to achieve an oral tolerance, butthe effect is in most cases not significant. In any event, the resultsare not sufficient for the methods to be translated to humans. The majorproblem in all these experiments is that no active suppression is beingobserved through the induction of CD4⁺ T-cells and subsequent productionof antigen specific regulatory T-cells. Only if this is being observed,a true and active suppression of immune response to antigens can beobtained in humans.

Targeted and more efficient delivery of molecules for therapeutic andprophylactic applications is a priority for the pharmaceutical industry.Effective strategies should reduce the required dose, increase safetyand improve efficacy by focusing molecules at the desired site ofaction. Mucosal routes of drug and vaccine delivery offer a number oflogistical and biological advantages compared with injection. Oraldelivery is particularly attractive as a result of the ease ofadministration. However, gastrointestinal degradation and low levels ofabsorption generally render this route of peptide and protein drugdelivery ineffective. Alternative mucosal routes such as the nasal,rectal, pulmonary and ocular routes are also being investigated. Mucosaldelivery of protein and peptide vaccine antigens generally stimulatespoor immune responses and may induce immunological tolerance.

Mucosal delivery of IL-4, TGF-β, IL-10 (Slavin et al., 2001) andanti-IL-12 have all been hypothesized to enhance tolerance.Interleukin-10 (IL-10) plays a critical role in the development oflow-dose tolerance (Slavin et al., 2001; Mauer et al., 2003). It hasbeen shown that treatment of mice with low-dose oral myelin basicprotein and simultaneous oral IL-10 reduces the severity and incidenceof experimental autoimmune encephalomyelitis, but the therapeutic effectis low and far from sufficient to be effective in human. In theseexperiments the amount of IL-10 feeding is high (1 μg to 10 μg), whilethe figures suggest that administering higher doses is more effective.Although a suppressive effect was observed of 0.1 μg IL-10 in vitro onproliferation, IL-12 and IFN-γ secretion, no effect of 0.1 μg IL-10 plusMBP treatment was seen upon disease. The same mice experiments have beendone with oral administration of IL-10 combined with low-dose oralmyelin oligodendrocyte glycoprotein (MOG), which resulted in reducedrelapses in a MOG-induced mouse model. Also here the therapeutic effectis low and the amount of IL-10 feeding high, showing figures that usinghigher doses is more effective. In both experiments there is no, or atleast insufficient, active suppression of an immune response via along-lasting immune tolerance to be effective in humans. In particular,because to assert a real therapeutic effect, sufficient to be translatedto humans, an induction of antigen specific CD4⁺ T-cells should beobserved, finally resulting in a production of regulatory T-cells. Onlysuch mechanism will be able to actively suppress the immune response inhumans. In all of the aforementioned examples no induction of CD4⁺T-cells has been observed.

It is generally agreed that the microflora plays a role in the inductionof oral tolerance (Moreau and Corthier, 1988; Gaboriau-Routhiau et al.,2003). Di Giacinto et al. (2005) suggest that probiotics may induceIL-10 and IL-10-dependent TGF-β-bearing regulatory cells. However, howthis effect is exerted is far from clear, and the simple presence ofmicro-organisms in the gut is not sufficient (Rask et al., 2005).Moreover, although probiotics may improve the symptoms of allergy andasthma, the results are not always unambiguous and the use of probioticsalone is not sufficient to induce a reliable oral tolerance response.Several attempts have been made to deliver low-dose antigens via lacticacid bacteria to prevent an allergic immune response (Daniel et al.,2006) and which led to reduced allergen specific IgE and enhancedallergen-specific secretory IgA responses. Although a desired shift inthe immune balance from T helper-2 type response towards a more Thelper-1 response is being achieved in mouse, there are no significantimprovements over the delivery of free allergens. In general, such anapproach of a sole delivery of allergens will not be sufficient toachieve the same result in humans. This is due to the fact that suchstrategies will require a very long period of intermittent treatments,while an induction of regulatory T-cells is not achieved, or at leastnot sufficiently to install a regulatory compartment to achieve a true,active and long-lasting immune tolerance effect. In another example theoral administration of recombinant lactobacilli expressing myelinantigens resulted in a reduced experimental autoimmune encephalomyelitisin a mouse model (Maassen et al., 2003). However, the therapeutic effectis considered to be low and not sufficient to be translated to humans.In particular, because to assert a real therapeutic effect, sufficientto be translated to humans, an induction of antigen specific CD4⁺T-cells should be observed, finally resulting in a production ofregulatory T-cells. Only such mechanism will be able to activelysuppress the immune response in humans. In all of the aforementionedexamples no induction of CD4⁺ T-cells has been examined.

Thus, there remains a problem in the art to effectively induce toleranceof antigens.

SUMMARY OF THE INVENTION

Surprisingly, we found that the mucosal delivery of an antigen, incombination of the mucosal delivery of micro-organism producing animmuno-modulating compound can induce a stable mucosal toleranceresponse, preferably if such antigen is expressed by a micro-organismand preferably if such mucosal delivery is done orally. We observed thatthe mucosal delivery of such combination gives a significantly bettersuppression of the antigen-specific immune response in comparison to thesole mucosal delivery of antigen expressing micro-organism. Even moresurprisingly, the immune suppression obtained through the invention issignificantly more effective than compared to oral delivery of freeimmuno-modulating compounds whether or in combination with the oraldelivery of antigens.

We demonstrate that the invention can induce oral tolerance with muchmore higher efficiency than with monotherapy with antigen or IL-10producing L. lactis alone, or than antigen combined with free orallyadministered IL-10. In vivo activation of antigen-specific regulatory Tcells was strongly enhanced. These cells transfer dominant tolerance toimmuno-competent recipients and mediate even bystander suppression. Theefficacy of the invention was demonstrated in autoimmune and allergicdisease mouse models, as well as in the context of immune inactivationof therapeutics.

DETAILED DESCRIPTION OF THE INVENTION

Throughout this disclosure, various publications, patents and publishedpatent specifications are referenced by an identifying citation. Thedisclosures of these publications, patents and published patentspecifications are hereby incorporated by reference into the presentdisclosure to describe more fully the state of the art to which thisinvention pertains.

A. General Techniques

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of organic chemistry, pharmacology,molecular biology (including recombinant techniques), cell biology,biochemistry, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature, such as,“Molecular Cloning: A Laboratory Manual” Second Edition (Sambrook etal., 1989); “Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “AnimalCell Culture” (R. I. Freshney, ed., 1987); the series “Methods inEnzymology” (Academic Press, Inc.); “Handbook of ExperimentalImmunology” (D. M. Weir & C. C. Blackwell, eds.); “Gene Transfer Vectorsfor Mammalian Cells” (J. M. Miller & M. P. Calos, eds., 1987); “CurrentProtocols in Molecular Biology” (F. M. Ausubel et al., eds., 1987, andperiodicals) “Polymerase Chain Reaction” (Mullis et al., eds., 1994);and “Current Protocols in Immunology” (J. E. Coligan et al., eds.,1991).

B. Definitions

As used herein, certain terms may have the following defined meanings.As used in the specification and claims, the singular forms “a”, “an”and “the” include plural references unless the context clearly dictatesotherwise. For example, the term “a cell” includes a plurality of cells,including mixtures thereof. Similarly, use of “a compound” for treatmentor preparation of medicaments as described herein contemplates using oneor more compounds of this invention for such treatment or preparationunless the context clearly dictates otherwise.

As used herein, the term “comprising” is intended to mean that thecompositions and methods include the recited elements, but not excludingothers. “Consisting essentially of” when used to define compositions andmethods, shall mean excluding other elements of any essentialsignificance to the combination. Thus, a composition consistingessentially of the elements as defined herein would not exclude tracecontaminants from the isolation and purification method andpharmaceutically acceptable carriers, such as phosphate buffered saline,preservatives, and the like.

“Consisting of” shall mean excluding more than trace elements of otheringredients and substantial method steps for administering thecompositions of this invention.

Embodiments defined by each of these transition terms are within thescope of this invention.

A first aspect of the invention is a method for inducing immunetolerance to an antigen, comprising mucosal delivery of said antigen, incombination with mucosal delivery of an immuno-modulating compoundproducing micro-organism.

Preferably, the present invention relates to the use of animmuno-modulating compound producing micro-organism in combination withan antigen for the preparation of a medicament for mucosal delivery toinduce immune tolerance.

Preferably, said immune tolerance is induced in an animal. Said animalis a mammal, and preferably chosen from the group consisting of mouse,rat, pig, cow, sheep, horses and human. Preferably, said mammal ishuman.

Preferably, said immune tolerance is mucosal tolerance.

Mucosa as used here can be any mucosa such as oral mucosa, rectalmucosa, urethral mucosa, vaginal mucosa, ocular mucosa, buccal mucosa,pulmonary mucosa and nasal mucosa. Mucosal delivery as used throughoutthe application encompasses the delivery to the mucosa. Oral mucosaldelivery includes buccal, sublingual and gingival routes of delivery.Accordingly, the present invention relates to method in which saidmucosal delivery is chosen from the group consisting of rectal delivery,buccal delivery, pulmonary delivery, ocular delivery, nasal delivery,vaginal delivery and oral delivery. Preferably, said mucosal delivery isoral delivery and said tolerance is oral tolerance.

Mucosal tolerance as used here throughout the application is theinhibition of specific immune responsiveness to an antigen in an animal(including humans), after that said animal has been exposed to saidantigen via the mucosal route. Preferably, said mucosal tolerance issystemic tolerance. The subsequent exposure of the antigen can be everyexposure known to the person skilled in the art, such as exposure byparenteral injection, by mucosal delivery, or by endogenous productionsuch as in the case of auto-antigens. Oral tolerance is the inhibitionof specific immune responsiveness to an antigen in an animal (includinghumans), after that said animal has been exposed to said antigen via theoral route. Low dose oral tolerance is oral tolerance induced by lowdoses of antigens, and is characterized by active immune suppression,mediated by cyclophosphamide sensitive regulatory T-cells that cantransfer tolerance to naïve hosts. High dose oral tolerance is oraltolerance induced by high doses of antigens, is insensitive tocyclophosphamide treatment, and proceeds to induction of T cellhyporesponsiveness via anergy and/or deletion of antigen specificT-cells. The difference in sensitivity to cyclophosphamide can be usedto make a distinction between low dose and high dose tolerance (Strobelet al., 1983). Preferably, said oral tolerance is low dose oraltolerance as described by Mayer and Shao (2004b).

The present invention thus relates to a method or use as describedherein, wherein said induction of immune tolerance is at least 1.5,preferably 2, more preferably 3 times or more relative to before saidinduction. Alternatively, said antigen is tolerated at least 1.5, 2, 3times or more relative to before said induction. The induction of immunetolerance can be measured by methods known in the art. Preferably, saidinduction of immune tolerance can be measured by modulation of acytokine level in said animal. As such, the modulation can be anincrease of a cytokine level, for instance said increase of a cytokinelevel is at least 1.5, 2, 3 times or more relative to before saidinduction. Alternatively, said modulation is a decrease of the level ofa particular cytokine level, for instance said decrease of the cytokinelevel is at least 1.5, 2, 3 times or more relative to before saidinduction. The cytokines may be chosen from any relevant cytokines,preferably said cytokines are chosen from the group consisting of IL-2,IL-4, IL-6, IL-10, IL-12, TNF-α, IFN-γ, IFN-α, MCP-1, TGFβ, RANK-L andFlt3L.

An antigen can be any antigen known to the person skilled in the art. Anantigen as used here throughout the application is preferably anysubstance that provokes an immune response when introduced in the bodyof an animal, wherein said immune response can be T-cell mediated and/ora B-cell mediated response. T-cell mediated responses cover Th1 and/orTh2 responses. The antigen can be any antigen, such as, but not limitedto allergens (including food allergens), allo-antigens, self-antigens,auto-antigens, and therapeutic molecules or antigens that induce animmune response. Preferably, said antigen is involved in the inductionof immune response related diseases. Even more preferably, said antigenis involved in the induction of allergic asthma, multiple sclerosis,type I diabetes, autoimmune uveitis, autoimmune thyroiditis, autoimmunemyasthenia gravis, rheumatoid arthritis, food allergy, celiac disease orgraft versus host disease.

An immune response related disease as used here is a disease caused byan unwanted immune response of the body against an antigen, whereby saidantigen can be either a heterologous antigen or an auto-antigen. Immuneresponse related diseases include, but are not limited to allergicreaction including food allergy, celiac disease, allergic asthma,autoimmune uveitis, autoimmune thyroiditis, autoimmune myastheniagravis, rheumatoid arthritis, type I diabetes and multiple sclerosis.Immune response related diseases also include unwanted immune reactionssuch as graft versus host disease or immuno-activation of medicationsuch as the antibody production against non endogenous Factor VIII.Preferably, the disease is selected from the group consisting ofallergic asthma, food allergy, celiac disease, type I diabetes andimmune inactivation of therapeutics. It will thus be appreciated thatimmune response related diseases include, but are not limited toallergic reaction including food allergy, celiac disease, allergicasthma, autoimmune uveitis, autoimmune thyroiditis, autoimmunemyasthenia gravis, rheumatoid arthritis, type I diabetes and multiplesclerosis. Immune response related diseases also include unwanted immunereactions such as graft versus host disease or immuno-activation ofmedication such as the antibody production against non endogenous FactorVIII. Preferably, the disease is selected from the group consisting ofallergic asthma, food allergy, celiac disease, graft versus hostdisease, type I diabetes and immune inactivation of therapeutics.

In further embodiments, said antigen is delivered by an antigenexpressing micro-organism. Preferably said antigen is delivered by anantigen secreting or antigen displaying micro-organism. Thus, theinvention relates to a method as described herein wherein said antigenis displayed at the surface of said antigen expressing micro-organism orwherein said antigen is expressed. The immuno-modulating compound andthe antigen may be delivered by the same micro-organism, or it may be adifferent micro-organism.

In view of the above, it will thus be appreciated that the presentinvention relates to method or use as described herein, wherein saidmethod or use is therapeutic and/or prophylactic.

Compound means any chemical of biological compound or complex, includingsimple or complex organic and inorganic molecules, peptides,peptido-mimetics, proteins, protein complexes, antibodies,carbohydrates, nucleic acids or derivatives thereof. Animmuno-modulating compound is a compound that modifies the function ofthe immune system. An immuno-modulating compound as used here is atolerance inducing compound; tolerance induction can be obtained, as anon-limiting example, in a direct way by inducing regulatory T-cellssuch as Treg, Tr1 or Th3, or by shifting the Th1/Th2 balance towardsTh1, or in an indirect way, by activation of immature dendritic cells totolerizing dendritic cells and/or inhibiting Th2 immune responseinducing expression of “co-stimulation” factors on mature dendriticcells. Immuno-modulating and immuno-suppressing compounds are known tothe person skilled in the art and include, but are not limited tobacterial metabolites such as spergualin, fungal and streptomycalmetabolites such as tacrolimus or cyclosporin, immuno-suppressingcytokines such as IL-4, IL-10, IFNα TGFβ (as selective adjuvant forregulatory T-cells) Flt3L, TSLP and Rank-L (as selective tolerogenic DCinducers), antibodies and/or antagonist such as anti-CD40L, anti-CD25,anti-CD20, anti-IgE, anti-CD3 and proteins, peptides or fusion proteinssuch as the CTL-4 Ig or CTLA-4 agonist fusion protein.

Thus, the immuno-modulating compound can be any immuno-modulatingcompound known to the person skilled in the art. Preferably, saidimmuno-modulating compound is an immuno-suppressing compound, even morepreferably said compound is an immuno-suppressing cytokine or antibody.Preferably, said immuno-suppressing cytokine is a tolerance-enhancingcytokine or antibody. Immuno-suppressing cytokines are known to theperson skilled in the art, and include, but are not limited to IL-4,IL-10, IFN-α and TGFβ, as selective adjuvant for regulatory T-cells; andFlt3L, TSLP and Rank-L, as selective tolerogenic DC inducers.Preferably, said immuno-suppressing cytokine is selected from the groupconsisting of IL-4, IL-10, IFNα and Flt3L. It will be appreciated by theperson skilled in the art that the present invention also relates tofunctional homologues thereof. A functional homologue connotes amolecule having essentially the same or similar, at least for theintended purposes, function, but can differ structurally. Mostpreferably, said immuno-suppressing tolerance-enhancing cytokine isIL-10, or a functional homologue thereof. Preferably, saidimmuno-suppressing antibody is chosen from the group consisting ofanti-IL-2, anti-IL12, anti-IL6 anti-IFN-γ.

Delivery as used here means any method of delivery known to the personskilled in the art and Includes, but is not limited to, coated ornon-coated pharmaceutical formulations of the compound to deliver,capsules, liposomes, oil bodies, polymer particles comprising orcarrying the compound to deliver or micro-organisms secreting,displaying or accumulating the compound to deliver, optionally inpresence of compounds that may enhance mucosal delivery and/or mucosaluptake.

Compounds or compositions described herein may be administered in pureform, combined with other active ingredients, or combined withpharmaceutically acceptable nontoxic excipients or carriers. Oralcompositions will generally include an inert diluent carrier or anedible carrier. Pharmaceutically compatible binding agents, and/oradjuvant materials can be included as part of the composition. Tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a dispersing agent such as alginic acid, Primogel,or corn starch; a lubricant such as magnesium stearate; a glidant suchas colloidal silicon dioxide; a sweetening agent such as sucrose orsaccharin; or a flavoring agent such as peppermint, methyl salicylate,or orange flavoring. When the dosage unit form is a capsule, it cancontain, in addition to material of the above type, a liquid carriersuch as a fatty oil. In addition, dosage unit forms can contain variousother materials that modify the physical form of the dosage unit, forexample, coatings of sugar, shellac, or enteric agents. Further, a syrupmay contain, in addition to the active compounds, sucrose as asweetening agent and certain preservatives, dyes, colorings, andflavorings. It will be appreciated that the form and character of thepharmaceutically acceptable carrier is dictated by the amount of activeingredient with which it is to be combined, the route of administrationand other well-known variables. The carrier(s) must be “acceptable” inthe sense of being compatible with the other ingredients of theformulation and not deleterious to the recipient thereof.

Alternative preparations for administration include sterile aqueous ornonaqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are dimethylsulfoxide, alcohols, propylene glycol,polyethylene glycol, vegetable oils such as olive oil and injectableorganic esters such as ethyl oleate. Aqueous carriers include mixturesof alcohols and water, buffered media, and saline. Intravenous vehiclesinclude fluid and nutrient replenishers, electrolyte replenishers, suchas those based on Ringer's dextrose, and the like. Preservatives andother additives may also be present such as, for example, antimicrobialsanti-oxidants, chelating agents, inert gases, and the like. Variousliquid formulations are possible for these delivery methods, includingsaline, alcohol, DMSO, and water based solutions.

Preferably said immuno-suppressing cytokine is expressed in low amounts,preferably 0.1 μg or lower per dose bacteria administered in a miceexperimental setting, such amounts to be translated in a human diseasesetting. We demonstrate that the invention can induce oral tolerancewith much more higher efficiency than with monotherapy with antigen orIL-10 producing micro-organism, such as L. lactis alone, or than antigencombined with free orally administered IL-10. In vivo activation ofantigen-specific regulatory T cells was strongly enhanced. These cellstransfer dominant tolerance to immuno-competent recipients and mediateeven bystander suppression. The efficacy of the invention wasdemonstrated in autoimmune and allergic disease mouse models, as well asin the context of immune inactivation of therapeutics.

The terms “treatment”, “treating”, and the like, as used herein includeamelioration or elimination of a developed mental disease or conditiononce it has been established or alleviation of the characteristicsymptoms of such disease or condition. As used herein these terms alsoencompass, depending on the condition of the patient, preventing theonset of a disease or condition or of symptoms associated with a diseaseor condition, including reducing the severity of a disease or conditionor symptoms associated therewith prior to affliction with said diseaseor condition. Such prevention or reduction prior to affliction refers toadministration of the compound or composition of the invention to apatient that is not at the time of administration afflicted with thedisease or condition. “Preventing” also encompasses preventing therecurrence or relapse-prevention of a disease or condition or ofsymptoms associated therewith, for instance after a period ofimprovement. It should be clear that mental conditions may beresponsible for physical complaints. In this respect, the term“treating” also includes prevention of a physical disease or conditionor amelioration or elimination of the developed physical disease orcondition once it has been established or alleviation of thecharacteristic symptoms of such conditions.

As used herein, the term “medicament” also encompasses the terms “drug”,“therapeutic”, “potion” or other terms which are used in the field ofmedicine to indicate a preparation with therapeutic or prophylacticeffect.

It will be appreciated that the compounds of the invention, i.e. theantigen and the immuno-modulating molecule are delivered or expressed ina therapeutically effective amount. As used herein, the term“therapeutically effective amount” is meant to refer to an amount of acompound or composition of the present invention that will elicit adesired therapeutic or prophylactic effect or response when administeredaccording to the desired treatment regimen. Preferably the compounds orcomposition is provided in a unit dosage form, for example a tablet,capsule or metered aerosol dose, so that a single dose is administeredto the subject, e.g. a patient.

In combination with, as used her throughout the application implies, ata certain moment, the simultaneous presence of the antigen and theimmuno-modulating compound at the level of the mucosa. It does not implythat both antigen and immuno-modulating compound always need to bepresent simultaneously at mucosal level. Therefore, the method coversboth simultaneous administration of antigen and immuno-modulatingcompound producing micro-organisms, as well a by sequentialadministration of antigen and immuno-modulating compound producingmicro-organism, or any combination thereof.

In a further embodiment, said antigen is delivered simultaneously withor sequential to said immuno-modulating compound secretingmicro-organism.

A preferred embodiment is simultaneous administration of antigen andimmuno-modulating compound producing micro-organism. In this case,antigen and immuno-modulating compound producing micro-organism may becomprised in the same pharmaceutical formulation, or in more than onepharmaceutical formulation taken together. A preferred embodiment isdelivery by a micro-organism producing both the antigen and theimmuno-modulating compound.

When the antigen and the immuno-modulating compound expressingmicro-organism or the composition comprising both elements areadministered simultaneously, the compounds or active ingredients may bepresent in a single pharmaceutical composition or formulation.

Alternatively the compounds or active ingredients are administered inseparate pharmaceutical compositions or formulations for simultaneous orseparate use. The invention thus also relates to pharmaceuticalcompositions comprising antigen and the immuno-modulating moleculeexpressing micro-organism of the invention and to the uses of thesepharmaceutical compositions.

In case of sequential administration, either the antigen or theimmunomodulating compound producing micro-organism may be administeredfirst. In case of sequential administration, the time between theadministration or the antigen and the immuno-modulating compoundproducing micro-organism is preferably not more than 3 hours, even morepreferably not more than two hours, most preferably not more than onehour.

The active ingredients may be administered from 1 to 6 times a day,sufficient to exhibit the desired activity. These daily doses can begiven as a single dose once daily, or can be given as two or moresmaller doses at the same or different times of the day which in totalgive the specified daily dose. Preferably, the active ingredient isadministered once or twice a day. It is contemplated that both activeagents would be administered at the same time, or very close in time.Alternatively, one compound could be taken in the morning and one laterin the day. Or in another scenario, one compound could be taken twicedaily and the other once daily, either at the same time as one of thetwice-a-day dosing occurred, or separately. Preferably both compoundswould be taken together at the same time and be administered as anadmixture. In an embodiment, the second compound is administeredsimultaneously with, separate from or sequential to said first compound.

In all aspects of the invention, the daily maintenance dose can be givenfor a period clinically desirable in the patient, for example from 1 dayup to several years (e.g. for the mammal's entire remaining life); forexample from about (2 or 3 or 5 days, 1 or 2 weeks, or 1 month) upwardsand/or for example up to about (5 years, 1 year, 6 months, 1 month, 1week, or 3 or 5 days). Administration of the daily maintenance dose forabout 3 to about 5 days or for about 1 week to about 1 year is typical.Other constituents of the liquid formulations may include preservatives,inorganic salts, acids, bases, buffers, nutrients, vitamins, or otherpharmaceuticals.

The micro-organism secreting the immuno-modulating compound and/or theantigen may be delivered in a dose of at least 10⁴ colony forming units(cfu) to 10¹² cfu per day, preferably between 10⁶ cfu to 10¹² cfu perday, most preferably between 10⁹ cfu and 10¹² cfu per day. In accordancewith the method as described in Steidler et al. (Science 2000), theimmuno-modulating compound of e.g. of 10⁹ cfu is secreted to at least 1ng to 100 ng. Through ELISA, known to a person skilled in the art, theantigen of e.g. of 10⁹ cfu is secreted to at least 1 ng to 100 ng; theskilled person in the art can calculate the range of secretion ofimmuno-modulating compound and/or antigen in relation to any other doseof cfu.

The antigen may be delivered in dose inducing a low-dose response.Preferably, said antigen is delivered in a dose of at least 10 fg to 100μg per day, preferably between 1 μg and 100 μg per day, most preferablybetween 1 ng and 100 μg per day.

The immuno-modulating compound of the invention may be delivered in adose of at least 10 fg to 100 μg per day, preferably between 1 μg and100 μg per day, most preferably between 1 ng and 100 μg per day.

Preferably the compounds or composition is provided in a unit dosageform, for example a tablet, solution, capsule or metered aerosol dose,so that a single dose is administered to the subject, e.g. a patient.

Depending on the mode of administration, e.g. oral, or any of the onesdescribed above, the man skilled in the art knows how to define orcalculate the actual dose to be administered to a patient. The personskilled in the art will be knowledgeable to adjust the doses dependingon the patient, micro-organism, vector et cetera.

Compounds of the present invention also may take the form of apharmacologically acceptable salt, hydrate, solvate, or metabolite.Pharmacologically acceptable salts include basic salts of inorganic andorganic acids, including but not limited to hydrochloric acid,hydrobromic acid, sulphuric acid, phosphoric acid, nitric acid,methanesulphonic acid, ethanesulfonic acid, p-toluenesulfonic acid,naphthalenesulfonic acid, malic acid, acetic acid, oxalic acid, tartaricacid, citric acid, lactic acid, fumaric acid, succinic acid, maleicacid, salicylic acid, benzoic acid, phenylacetic acid, mandelic acid andthe like. When compounds of the invention include an acidic function,such as a carboxy group, then suitable pharmaceutically acceptablecation pairs for the carboxy group are well known to those skilled inthe art and include alkaline, alkaline earth, ammonium, quaternaryammonium cations and the like.

The micro-organism can be any micro-organism, including bacteria, yeastsor fungi, suitable for mucosal delivery. Preferably, said micro-organismis a non pathogenic micro-organism, even more preferably saidmicro-organism is a probiotic micro-organism. Probiotic organisms areknown to the person skilled in the art. Probiotic organisms include, butare not limited to, bacteria such as Lactobacillus sp., Lactococcus sp.,Bifidobacterium sp. and yeasts such as Saccharomyces cerevisiaesubspecies boulardii. Preferably, said bacterium is a lactic acidbacterium; Even more preferably, said lactic acid bacterium is chosenfrom the group consisting of Lactobacillus, Leuconostoc, Pediococcus,Lactococcus, Streptococcus, Aerococcus, Camobacterium, Enterococcus,Oenococcus, Teragenococcus, Vagococcus, and Weisella. In one furtherpreferred embodiment, said micro-organism is Lactococcus lactis. Inanother preferred embodiment, said micro-organism is Saccharomycescerevisiae.

In a preferred embodiment, the immuno-suppressing cytokine is combinedwith antagonizing antibodies against immuno-inducing cytokines, such asanti-IL-2, anti-IL-12 and/or anti-IFNγ; and costimulatory molecules,such as anti-CD40L and anti-CD3. Alternatively, compounds may bedelivered that stimulate the production of the immuno-suppressingcytokines, such as cholera toxin B subunit; and molecules that stimulateregulatory T cell function, such ICOS and CTLA-4 agonists. As describedabove, preferably, said micro-organism is a non-pathogenicmicro-organism, even more preferably it is a probiotic micro-organism.Probiotic organisms are known to the person skilled in the art, andinclude, but are not limited to bacteria such as Lactobacillus sp.,Lactococcus sp., Bifidobacterium sp. and yeasts such as Saccharomycescerevisiae subspecies boulardii. In one preferred embodiment, saidmicro-organism is Lactococcus lactis. In another preferred embodiment,said micro-organism is Saccharomyces cerevisiae. Most preferably saidprobiotic micro-organism is a lactic acid bacterium, as delivery ofheterologous proteins (i.e. non Lactic acid bacterial proteins) bylactic acid bacteria into the mucosa, including both oral and vaginaldelivery, has been described (Steidler and Rottiers, 2006; Liu et al.,2006), which makes these lactic acid bacteria extremely suitable fordelivery of both antigen and immuno-suppressing compound.

Another aspect of the invention is the use of an immuno-modulatingcompound producing micro-organism, in combination of an antigen for thepreparation of a medicament to treat an immune response related disease.Preferably, said immuno-modulating compound is an immuno-suppressingcytokine. Preferably, said antigen is delivered by an antigen secretingmicro-organism. The immuno-modulating compound and the antigen may bedelivered by the same micro-organism, or it may be a differentmicro-organism. Preferably, said immuno-suppressing cytokine is aimmuno-suppressing, tolerance-enhancing cytokine. Immuno-suppressing,tolerance-enhancing cytokines are known to the person skilled in theart, and include, but are not limited to IL-4, IL-10, IFNα and TGFβ,Flt3L and Rank-L. Preferably, said immuno-suppressing cytokine isselected from the group consisting of IL-4, IL-10, IFNα and Flt3L. Mostpreferably, said immuno-suppressing cytokine is IL-10, or a functionalhomologue thereof. In one preferred embodiment, the immuno-suppressingcytokine is combined with antagonizing antibodies againstimmuno-inducing cytokines, such as anti-IL-2, anti-IL-12 and/or antiIFNγ and costimulatory molecules, such as anti-CD40L and anti-CD3.Preferably said immuno-suppressing cytokine is expressed in low amounts,preferably 0.1 μg or lower in a mice experimental setting, such amountsto be translated in a human disease setting.

It will be appreciated that the compounds and compositions of theinvention may be used as nutraceuticals, functional or medical food, oras additives in said nutraceuticals, functional or medical food. Anotherembodiment provides a food or beverage, preferably fit for humanconsumption, which is comprised of a nutraceutical and a flavoringagent, wherein the nutraceutical is comprised of an extract from anagricultural product.

Nutraceuticals, whether in the form of a liquid extract or drycomposition, are edible and may be eaten directly by humans, but arepreferably provided to humans in the form of additives or nutritionalsupplements e.g., in the form of tablets of the kind sold in health foodstores, or as ingredients in edible solids, more preferably processedfood products such as cereals, breads, tofu, cookies, ice cream, cakes,potato chips, pretzels, cheese, etc., and in drinkable liquids e.g.,beverages such as milk, soda, sports drinks, and fruit juices. Thus, inone embodiment a method is provided for enhancing the nutritional valueof a food or beverage by intermixing the food or beverage with anutraceutical in an amount that is effective to enhance the nutritionalvalue of the food or beverage.

Another embodiment provides a method for enhancing the nutritional valueof a food or beverage which comprises intermixing a food or a beveragewith a nutraceutical to produce a nutritionally-enhanced food orbeverage, wherein the nutraceutical is intermixed in an amount effectiveto enhance the nutritional value of the food or beverage, wherein thenutraceutical is comprised of an extract from a crop comprising theantigens of the present invention, and wherein thenutritionally-enhanced food or beverage may further comprise a flavoringagent. Preferred flavoring agents include sweeteners such as sugar, cornsyrup, fructose, dextrose, maltodextrose, cyclamates, saccharin,phenyl-alanine, xylitol, sorbitol, maltitol, and herbal sweeteners e.g.,Stevia.

The nutraceuticals described herein are intended for human consumptionand thus the processes for obtaining them are preferably conducted inaccordance with Good Manufacturing Practices (GMP) and any applicablegovernment regulations governing such processes. Especially preferredprocesses utilize only naturally derived solvents. The nutraceuticalsdescribed herein preferably contain relatively high levels ofhealth-enhancing substances Nutraceuticals may be intermixed with oneanother to increase their health-enhancing effects.

In contrast to nutraceuticals, the so-called “medical foods” are notmeant to be used by the general public and are not available in storesor supermarkets. Medical foods are not those foods included within ahealthy diet to decrease the risk of disease, such as reduced-fat foodsor low-sodium foods, nor are they weight loss products. A physicianprescribes a medical food when a patient has special nutrient needs inorder to manage a disease or health condition, and the patient is underthe physician's ongoing care. The label must clearly state that theproduct is intended to be used to manage a specific medical disorder orcondition. An example of a medical food is nutritionally diverse medicalfood designed to provide targeted nutritional support for patients withchronic inflammatory conditions. Active compounds of this product arefor instance one or more of the compounds described herein. Functionalfoods may encompass those foods included within a healthy diet todecrease the risk of disease, such as reduced-fat foods or low-sodiumfoods, or weight loss products. Hence, the present inventioncontemplates a food or beverage comprising a nutraceutical according tothe invention.

The present invention thus relates to the use of an immuno-modulatingcompound secreting micro-organism in combination with an antigen for thepreparation of a medicament, medical food or nutraceutical to induceimmune tolerance or to treat an immune response related disease.Preferably, the present invention relates to the use of a compositionfor the preparation and/or the manufacturing of a medicament, medicalfood or nutraceutical for treating, preventing and/or alleviating adisease or disorder involving an immune response related disease,characterized in that said composition comprises at least animmuno-modulating compound secreting micro-organism and an antigen.

In a further aspect, the present invention relates to the use of atleast an immuno-modulating compound secreting micro-organism and anantigen for treating, preventing and/or alleviating a disease ordisorder involving an immune response related disease. Hence, thepresent invention also relates to a method for treating an immuneresponse related disease in an animal in need thereof, comprisingmucosal delivery of an antigen in combination with mucosal delivery ofan immuno-modulating compound secreting micro-organism.

In a further embodiment the invention relates to a compositioncomprising an immuno-modulating compound secreting micro-organism incombination with an antigen. Preferably, said composition is apharmaceutical composition. Preferably, said antigen is an allergen,allo-antigen, self antigen or auto-antigen. Even more preferably, saidantigen is involved in the induction of allergic asthma, multiplesclerosis, type I diabetes, autoimmune uveitis, autoimmune thyroiditis,autoimmune myasthenia gravis, rheumatoid arthritis, food allergy orceliac disease. In one preferred embodiment, said antigen is atherapeutic antigen, preferably Anti-CD3. Preferably, the antigenaccording to the invention is delivered by an antigen expressingmicro-organism. In this case, the antigen may be displayed at thesurface of said antigen expressing micro-organism or it may be secretedby said organism. Preferably, said composition is presented in a spray,capsule, aerosol, lozenges, bolus, tablet, sachets, liquid, suspension,emulsion or troches, preferably in a unit dosage form, for example atablet, capsule or metered aerosol dose. Preferably, theimmuno-modulating compound of the composition according to the inventionis an immuno-suppressing compound or antibody. In one preferredembodiment said immuno-suppressing compound is a tolerance enhancingcytokine or a tolerance enhancing antibody, most preferably it is chosenfrom the group consisting of IL-4, IL10, IFN-α, Flt3L, TGFβ and RANK-L.In another preferred embodiment said immuno-suppressing compound is animmuno-suppressing antibody is chosen from the group consisting ofanti-IL-2, anti-IL12 and anti-IFN-γ. In one preferred embodiment, theimmuno-modulating compound secreting micro-organism of the compositionaccording to the invention is a probiotic micro-organism. In anotherpreferred embodiment, the immuno-modulating compound secretingmicro-organism of the composition according to the invention is abacterium or a yeast, preferably said bacterium is a lactic acidbacterium, even more preferably it is a lactic acid bacterium chosenfrom the group consisting of Lactobacillus, Leuconostoc, Pediococcus,Lactococcus, Streptococcus, Aerococcus, Camobacterium, Enterococcus,Oenococcus, Teragenococcus, Vagococcus, and Weisella, most preferablysaid Lactococcus is Lactococcus lactis. Preferably, said yeast isSaccharomyces cerevisiae. Preferably, said antigen and saidimmuno-modulating compound of the composition according to the inventionare expressed by the same micro-organism. Preferably the compositionaccording to the invention is further comprising an adjuvant,pharmaceutical acceptable carrier and/or excipient. Preferably, thecomposition according to the invention further comprises a compoundstimulating production of immuno-suppressing cytokines, preferably saidcompound stimulating production of immuno-suppressing cytokines ischolera toxin B subunit. Preferably, in the composition according to theinvention, said antigen and/or said immuno-modulating compound secretingmicro-organism are present in a dose of at least at least 10 femtogramto 100 mg.

In a final embodiment, the present invention relates to a medicament,nutraceutical or medical food for treating, preventing and/oralleviating a disease or disorder involving an immune response relateddisease comprising at least an antigen in combination with animmuno-modulating compound secreting micro-organism.

Those skilled in the art will appreciate that numerous changes andmodifications can be made to the preferred embodiments of the inventionand that such changes and modifications can be made without departingfrom the spirit of the invention. It is, therefore, intended that theappended claims cover all such equivalent variations as fall within thetrue spirit and scope of the invention.

In addition, all terms used in the description of compounds of thepresent invention have their meaning as is well known in the art.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Proliferative immune responses in the popliteal and inguinallymph nodes (PLN/ILN) following oral feeding with GM L. lactis orovalbumin protein (OVA) to Balb/c mice. OVA-specific proliferativeresponses were measured 11 days after subcutaneous challenge (on day 0)of the mice with OVA in complete Freund's adjuvant. Mice received mixedL. lactis suspension on days −46 till −42, −39 till −35, −32 till −28,−25 till −21, −18 till −14, −11 till −7, −4 till −1. LL-pT: mixedbacterial suspension of LL-pT1NX (vector control) and LL-pT1NX; LL-OVA:mixed bacterial suspension of L. lactis strain secreting ovalbumin andLL-pT1 NX; LL-OVA+LL-mIL10: mixed bacterial suspension of LL-OVA and L.lactis strain secreting murine interleukin-10. Positive control 1received 20 mg OVA on day −7. Positive control 2 received 1 μg OVA onthe same days as the L. lactis feeding. The results shown are the mean[³H]-thymidine incorporation in cpm (±SD) for triplicate cultures ofpooled cells from groups with 4 mice.

FIG. 2. Cytokine responses in the MLN and PLN/ILN following oral feedingwith GM L. lactis or OVA to Balb/c mice. The secretion of IL12p70 (a),TNF-α (b), IFN-γ (c), MCP-1 (d), IL-10 (e) and IL-6 (f) in control miceand mice fed GM L. lactis or OVA was evaluated. Cell culturesupernatants of MLN (A) and PLN/ILN (B) cells were tested followingrestimulation with 300 μg/ml OVA in vitro, for the presence of cytokinesby CBA (BD Bioscience), using the mouse inflammation kit. The resultsshown are the cytokine productions by pooled cells from groups with 4mice.

FIG. 3. OVA-specific proliferative CD4+ T-cell responses in the PLN/ILNfollowing oral feeding with GM L. lactis or ovalbumin protein (OVA) toBalb/c mice. OVA-specific proliferative responses were measured 11 daysafter subcutaneous challenge (on day 0) of the mice with OVA in completeFreund's adjuvant. Mice received mixed L. lactis suspension on days −46till −42, −39 till −35, −32 till −28, −25 till −21, −18 till −14, −11till −7, −4 till −1. LL-pT: mixed bacterial suspension of LL-pT1NX(vector control) and LL-pT1NX; LL-OVA: mixed bacterial suspension of L.lactis strain secreting ovalbumin and LL-pT1NX; LL-OVA+LL-mlL 10: mixedbacterial suspension of LL-OVA and L. lactis strain secreting murineinterleukin-10. Positive control 1 received 20 mg OVA on day −7.Positive control 2 received 1 μg OVA on the same days as the L. lactisfeeding. The results shown are the mean [³H]-thymidine incorporation incpm (±SD) for triplicate cultures of pooled cells from groups with 4mice.

EXAMPLES Example A Induction of Tolerance to Ovalbumin Following OralAdministration of L. lactis Secreting Said Ovalbumin in Combination withIn Situ Delivered IL-10 Material and Methods to the Examples Bacteriaand Plasmids

The L. lactis strain MG1363 was used throughout this study. Bacteriawere cultured in GM17 medium, i.e. M17 (Difco Laboratories, Detroit,Mich.) supplemented with 0.5% glucose. Stock suspensions of all strainswere stored at −20° C. in 50% glycerol in GM17. For intragastricinoculations, stock suspensions were diluted 500-fold in fresh GM17 andincubated at 30° C. They reached a saturation density of 2×10⁹colony-forming units (CFU) per ml within 16 hours. Throughout thisstudy, mixed bacterial suspensions were used. Therefore, the bacteriathat have to be mixed were harvested by centrifugation and pellets ofboth bacterial cultures were concentrated 10-fold in BM9 medium(Schotte, et al., 2000). For treatment, each mouse received 100 μl ofthis suspension by intragastric catheter.

The mRNA sequence encoding Gallus gallus Ovalbumin was retrieved fromGenbank (accession number AY223553). Total RNA was isolated from chickenuterus and cDNA was synthesized using 2 μg total RNA, 2 μM oligo dTprimers (Promega Corporation Benelux, Leiden, The Netherlands), 0.01 mMDTT (Sigma-Aldrich, Zwijndrecht, The Netherlands), 0.5 mM dNTP(Invitrogen, Merelbeke, Belgium), 20 U Rnasin (Promega IncorporationBenelux) and 100 U superscript II reverse transcriptase (Invitrogen) ina volume of 25 μl. OVA cDNA fragment was amplified by Polymerase ChainReaction (PCR) using the following conditions: 94° C. for 2 min followedby 30 cycles at 94° C. for 45 seconds, 62° C. for 30 seconds and 72° C.for 90 seconds, with the following forward and reverse primers

5′-GGCTCCATCGGTGCAGCAAGCATGGAATT-3′ and5′-ACTAGTTAAGGGGAAACACATCTGCCAAAGAAGAGAA-3′.

The amplified fragment was fused to the Usp45 secretion signal of theerythromycin resistant pT1NX vector, downstream of the lactococcal P1promoter. MG1363 strains transformed with plasmids carrying OVA cDNA andIL-10 were designated L. lactis secreting OVA (LL-OVA) and LL-IL10. TheL. lactis-pT1NX, which is MG1363 containing the empty vector, pT1NX,served as control (LL-pT1 NX).

Animals

7-week old female Balb/c mice were obtained from Charles RiverLaboratories (Italy). They were housed under SPF conditions and were fedstandard laboratory feed and tap water ad libitum. The animal's studieswere approved by the Ethics Committee of the Department for MolecularBiomedical Research, Ghent University.

Induction and Assessment of Oral Tolerance

Mice received mixed L. lactis suspension on days −46 till −42, −39 till−35, −32 till −28, −25 till −21, −18 till −14, −11 till −7, −4 till −1.LL-pT: mixed bacterial suspension of LL-pT1NX (vector control) andLL-pT1NX; LL-OVA: mixed bacterial suspension of L. lactis strainsecreting ovalbumin and LL-pT1NX; LL-OVA+LL-mlL 10: mixed bacterialsuspension of LL-OVA and L. lactis strain secreting murineinterleukin-10. Two positive controls for oral tolerance induction wereincluded in the study. Positive control 1 received 20 mg ovalbumin in100 μl BM9 medium on day −7. Positive control 2 received 1 μg ovalbuminin 100 μl BM9 medium on the same days as the L. lactis feeding. Micereceived feedings intragastrically by catheter. Control mice were notorally treated. On day 0, mice were immunized s.c. with 100 μg OVAemulsified 1:1 in complete Freund's adjuvant containing 100 μg M.tuberculosis H37 RA (Difco). Eleven days after the immunization,mesenteric lymph nodes (MLN) and popliteal and inguinal lymph nodes(PLN/ILN) were harvested and the cells assessed for OVA-specificproliferation and cytokine production.

OVA-Specific Proliferation In Vitro

Single cell suspension of the draining popliteal and inguinal lymphnodes were prepared. Cells were counted and resuspended at 2×10⁵ cellsin 200 μl RPMI-1640 containing 10% fetal calf serum (FCS), 10 U/mlpenicillin, 10 μg/ml streptomycin, 2 mM L-glutamax, 0.4 mM sodiumpyruvate (RPMI complete) either alone or with 11, 33, 100 or 300 μg/mlOVA. The cells were cultured for 90 hours in U-bottomed 96-well tissueculture plates (Becton Dickinson) at 37° C. in a 5% CO₂ humidifiedincubator. Proliferation was assessed by addition of 1 μCi/well[³H]-thymidine for the last 18 hours of culture. DNA-bound radioactivitywas harvested onto glass fiber filter mats (Perkin Elmer) andthymidine-incorporation measured on a scintillation counter (PerkinElmer).

OVA-Specific Proliferation of CD4 Purified T Cells In Vitro

CD4+ T-cells were purified from the whole cell preparations from thePLN/ILN using the CD4+ T-cell isolation kit (Miltenyi Biotec). 2×10⁵CD4+ T-cells were cultured in 200 μl RPMI complete with mitomycinC-treated splenocytes loaded with OVA, acting as antigen presentingcells at ratio's CD4+ T-cells/APC, 1/3, 1/1, 1/0.3, 1/0.1 and 1/0. Thecells were cultured for 90 hours in U-bottomed 96-well tissue cultureplates (Becton Dickinson) at 37° C. in a 5% CO₂ humidified incubator.Proliferation was assessed by addition of 1 μCi/well [³H]-thymidine forthe last 18 hours of culture. DNA-bound radioactivity was harvested ontoglass fiber filter mats (Perkin Elmer) and thymidine-incorporationmeasured on a scintillation counter (Perkin Elmer).

Measurement of OVA-Specific Cytokine Production

Lymph nodes cells from the mesenteric lymph nodes (MLN) and drainingpopliteal and inguinal lymph nodes were prepared and were resuspended at2×10⁶ cells/ml and 100 μl aliquots cultured in U-bottomed 96-well tissueculture plates for 72 hours with 300 μg/ml OVA. Supernatants were storedat −20° C. until cytokine levels were quantified by the Cytometric BeadArray using the mouse inflammation kit (BD Bioscience).

Example A1 LL-IL10 Significantly Enhances the Tolerance-InducingCapacity of LL-Ova

To study the induction of oral tolerance, mice were orally fed GM L.lactis [LL-pt: mixed bacterial suspension of LL-pT1NX [all] (=vectorcontrol) and LL-pT1NX; LL-OVA: mixed bacterial suspension ofOVA-secreting L. lactis [all italics] and LL-pT1NX; LL-OVA+LL-mIL-10:mixed bacterial suspension of OVA-secreting L. lactis and murine IL-10secreting L. lactis] 6 times 5 consecutive days (on days −46 till-42,−39 till −35, −32 till −28, −25 till −21, −18 till −14, −11 till −7 and−4 till −1) or a single dose of 20 mg OVA on day −7 [positive control 1]or frequent doses of 1 μg OVA on the same days as the L. lactis feeding[positive control 2]. Control mice were not orally treated. On day 0,mice were immunized s.c. with OVA in complete Freund's adjuvant andOVA-specific proliferation of the PLN/ILN cells was assessed on day 11.Addition of LL-IL-10 significantly enhanced the tolerance inductiontowards OVA as the OVA-specific proliferative response of the PLN/ILNcells (FIG. 1) was significantly reduced in the LL-OVA [all]+LL-mIL-10group in comparison to the control and LL-ova groups.

Example A2 LL-IL10 Potentiates Oral Tolerance in Association withReduced Production of Proinflammatory Cytokines in Response to Ova

To study the induction of oral tolerance, mice were orally fed GM L.lactis or OVA as described above (example 1) and were subsequentlyimmunized s.c. with OVA in complete Freund's adjuvant. Eleven daysfollowing the immunization, the cytokine production in response to OVAin the MLN and PLN/ILN was quantified by Cytometric Bead Array, usingthe mouse inflammation kit. In the MLN, the production of theproinflammatory cytokines, IL-12, TNF-α, IFN-γ and IL-6 was not detectedor strongly reduced in the LL-ova+LL-mIL-10 group in comparison to theLL-ova group in which a strong production of these pro-inflammatorycytokines was observed (FIG. 2A). In the PLN, the production of theproinflammatory cytokines, TNF-α, IFN-γ, MCP-1, and IL-6 is stronglyreduced in the LL-ova+LL-mIL-10 group in comparison to the LL-ova groupand in this group the TNF-α, MCP-1 and IL-6 levels are lower than thoseobserved in the control group (FIG. 2B).

Example A3 LL-IL10 Enhances Oral Tolerance Via CD4+ T Cells

To assess whether the induction of oral tolerance was mediated by CD4+[all] T cells, the OVA-specific proliferative CD4 T-cell response wasstudied in the MLN and PLN/ILN. Therefore, mice were orally fed with GML. lactis or OVA on the days indicated above (Example 1). The mice wereimmunized s.c. with OVA in complete Freund's adjuvant on day 0 and 11days later the CD4 T cells were purified from the MLN and PLN/ILN andwere subsequently cultured in presence of mitomycin C-treatedsplenocytes loaded with OVA. The OVA-specific CD4 T cell response in theLL-ova+LL-mIL-10 group was significantly reduced in comparison to theLL-ova and control groups (FIG. 3).

Example B Induction of Tolerance to Clotting Factor VIII and Factor IXFollowing Oral Administration of L. lactis Secreting Said Factors inCombination with In Situ Delivered IL-10 Introduction

Several therapeutic (recombinant) proteins, such as interferon's, factorVIII/IX and antibodies (Remicade) are administered at high doses overprolonged treatment periods. However, a complication associated withtheir use is the development of protein-specific immune responses, suchas antibodies. These antibodies (Abs), also called inhibitors, renderthe therapeutic proteins less effective. Examples include the formationof inhibitors for factor VIII/IX in hemophilia, erythropoietin (Epo) inpatients undergoing therapy for chronic renal failure, and IFN-β inpatients undergoing treatment for multiple sclerosis. Here, wedemonstrate that oral delivery of the Factor VIII (and Factor IX) incombination with IL-10 producing L. lactis suppresses inhibitorformation to said factor via the induction of antigen-specific CD4+regulatory T cells.

Material and Methods to the Examples Bacteria and Plasmids

The L. lactis strain MG1363 is used throughout this study. Bacteria arecultured in GM17 medium, i.e. M17 (Difco Laboratories, Detroit, Mich.)supplemented with 0.5% glucose. Stock suspensions of all strains arestored at −20° C. in 50% glycerol in GM17. For intragastricinoculations, stock suspensions are diluted 200-fold in fresh GM17 andare incubated at 30° C. They reach a saturation density of 2×10⁹colony-forming units (CFU) per ml within 16 hours. Throughout thisstudy, mixed bacterial suspensions are used. Therefore, the bacteriathat are mixed are harvested by centrifugation and pellets of bothbacterial cultures are concentrated 10-fold in BM9 medium (Schotte,Steidler et al. 2000). For treatment, each mouse receives 100 μl of thissuspension by intragastric catheter.

Human FVIII and FIX cDNA or cDNA-fragments, representing FVIII- andFIX-specific CD4+ T-cell epitopes, are amplified fused to the Usp45secretion signal of the erythromycin resistant pT1NX vector, downstreamof the lactococcal P1 promoter.

MG1363 strains transformed with plasmids carrying murine IL-10, FVIII(and/or epitope fragment), FIX (and/or epitope fragment), weredesignated L. lactis secreting IL10, LL-IL10, LL-FVIII, LL-FIX.LL-pT1NX, which is MG1363 containing the empty vector pT1NX, serve ascontrol.

Quantification of FVIII and FIX

FVIII or FIX from LL-FVIII and LL-IX, respectively are determined usinghuman FVIII and FIX-specific enzyme-linked immunosorbent assay (ELISA),that have been described previously (Chuah et al., 2003). Therecombinant proteins are also analyzed by Western blot analysis andCOATests and aPTT assays, as described (Chuah et al., 2003;VandenDriessche et al., 1999). The NH2-terminus of this protein isdetermined by automated Edman degradation. Since FVIII and FIX arenormally expressed in the liver where they undergo extensivepost-translational modifications, the clotting factors produced from theengineered L. lactis may be biologically inactive. However, thesepost-translational differences will likely have no repercussions on theability of these L. lactis-produced recombinant proteins to induceimmune tolerance. Indeed, most inhibitors that have been characterizedin detail to date typically recognize amino acid residues (Villard etal., 2003), rather than glycosylated moieties.

Animals

Hemophilia A or B mice obtained by knocking-out the murine FVIII or FIXgenes using homologous recombination in ES cells as described by (Bi etal., (1995) and Wang et al., (1997), are bred in the laboratory. Theserecipient mice generate neutralizing antibodies when challenged withpurified recombinant FVIII or FIX antigen in the presence of CFA(Mingozzi et al., 2003). The inhibitor status can be monitored over timeusing Bethesda assays or anti-FVIII/anti-FIX specific ELISAs. Recipientmice challenged with FVIII or FIX (+CFA) typically develop inhibitors2-3 weeks after antigenic challenge.

Experimental Setting

4-6 week-old mice receive LL-FVIII, LL-FIX, or LL-pT1NX or LL-OVA (anirrelevant antigen) either as negative controls, combined or not withLL-IL10 or IL-10 protein (1 or 10 μg). As a positive control fortolerance induction, we inject mice with adeno-associated viral vectors(AAV) expressing FIX from a hepatocyte-specific promoter. Recipientanimals develop FIX-specific immune tolerance that prevents induction ofanti-FIX antibodies upon subsequent challenge with FIX+CFA.

In a prophylactic setting, LL-FVIII, LL-FIX alone or along with LL-IL10or IL-10 are administered orally to hemophilia A or B mice using agastric catheter, using different treatment intervals and doses. Theserecipient mice are subsequently challenged with purified recombinantFVIII or FIX antigen, in the presence of CFA (Mingozzi et al., 2003).Control animals are exposed to LL-pT1NX and LL-OVA. Plasma is harvestedby retro-orbital bleeding. The development of antibodies directedagainst FVIII or FIX is assessed using Bethesda assays (Kasper et al.,1975) or using a modified anti-FVIII or anti-FIX specific ELISA(VandenDriessche et al., 1999) at different time intervals.

In a therapeutic setting, hemophilia A or B mice are first immunizedwith FVIII or FIX, as described (Mingozzi et al., 2003). The inhibitorstatus is monitored over time using Bethesda assays oranti-FVIII/anti-FIX specific ELISAs. Mice with low or high inhibitortiters are subsequently treated with LL-FVIII, LL-FIX alone or alongwith LL-IL10 or IL-10 using different treatment intervals and doses andinhibitor titers are determined over time. The specificity of thepossible immune tolerance is assessed by challenging the mice thatreceive LL-FVIII, LL-FIX alone or along with LL-IL10 with an irrelevantantigen (tetanus toxoid or Ova). As a positive control, mice are exposedorally to purified FVIII or FIX.

Cell Cultures, Proliferation and Cytokine Assay

Single cell suspensions of spleen and lymph nodes are prepared bypassing the cells through 70 μm filter cell strainers (Becton/DickinsonLabware). Erythrocytes are removed from the spleen cell suspensions byincubation with red cell lysis buffer.

Proliferation assays of total splenocyte populations, 2×10⁵ cells arecultured in 96-well U-bottom plates in a total volume of 200 μl completemedium either alone or with purified FVIII or FIX, and either with orwithout anti-IL-10 or anti-TGF-β neutralising monoclonal antibodies.FVIII and FIX is added at concentrations ranging from 1 to 100 μg/ml.The neutralizing antibodies are added at 1, 0.1 and 0.01 μg/ml. Forproliferation assays of CD4⁺ T cells and CD4⁺CD25⁻ T cell populations,0.2×10⁵ cells CD4⁺ T cells or CD4⁺ CD25⁻ T cells are cultured in 96-wellU-bottom plates with 1×10⁵ irradiated CD4⁻ cells, acting as antigenpresenting cells, and FVIII or FIX (0 or 100 μg/ml) in a total volume of200 μl complete medium either with or without neutralizing antibodies.After 72 hr at 37° C. in a 5% CO₂ humidified incubator, proliferation isassessed by addition of 1 μCi/well [³H]-thymidin. DNA-boundradioactivity is harvested 16-18 hr later onto glass fiber filter mats(Perkin Elmer, Boston, USA) and thymidine-incorporation is measured on ascintillation counter (Perkin Elmer).

For cytokine measurements, supernatants of the cell cultures used in thedifferent proliferation assays are collected after 24, 48 and 72 h ofculture and frozen at −20° C. until cytokine analysis is performed.Cytokine production is quantified using the Mouse InflammationCytometric Bead Assay (BD Biosciences, Mountain View, Calif., USA).

In Vivo T Regulatory Activity Assay

In order to test for active suppression of antibody formation in mice,splenocytes, bead-purified CD4⁺ T cells, CD4⁺ CD25⁻ or CD4⁺ CD25⁺ Tcells isolated from the different experimental L. Lactis-treated groupsare adoptively transferred to naïve C3H/HeJ mice. Untreated mice areused as control. The number of transferred cells is 10⁷ for whole spleencells, subpopulation-depleted spleen cells, or positively selected CD4⁺cells and CD4⁺ CD25⁻ and CD4⁺ CD25⁺ T cells. Recipient mice (n=4-5 perexperimental cohort) were subcutaneously injected with 5 μg hF.IX in cFA36 hours after adoptive transfer. Anti-hF.IX IgG titers in plasma weremeasured 2.5 weeks after immunization.

Example B1 LL-IL10 Significantly Enhances the Tolerance-InducingCapacity of LL-FVIII and LL-IX in Hemophilia A or B Mice

To study the induction of oral tolerance, mice are orally fed asdescribed above (experimental setting). Addition of LL-IL-10significantly enhances the tolerance induction towards FVIII and FIX asthe factor-specific proliferative response of splenocytes issignificantly reduced in the LL-FVIII/FIX+LL-mIL-10 group in comparisonto the control and LL-FVIII/IX groups.

Example B2 LL-IL10 Potentiates Oral Tolerance in Association withReduced FVIII- and FIX-Specific Titers and IFN-γ and More IL10 and TGF-βProduction in Response to Said Factor

To study the induction of oral tolerance, mice are orally fed asdescribed above (experimental setting). FVIII and FIX-specificantibodies and cytokine production in response to said factor insplenocytes and lymph nodes are quantified as described above. Theinhibitor formation and production of the proinflammatory cytokine,IFN-γ is strongly reduced and the immunosuppressive cytokines IL-10 andTGF-β is significantly increased in the LL-FVIII/FIX+LL-mIL-10 group incomparison to the control and LL-FVIII/IX groups.

Example B3 LL-IL10 Enhances Oral Tolerance Via CD4+ T Cells

To assess whether CD4+ T cells mediate the induction of oral tolerance,the factor-specific proliferative CD4+ T-cell response is studied in thesplenocytes and lymph nodes. Therefore, mice are orally fed as describedabove (experimental setting) and the factor-specific CD4+ T cellproliferation is determined as described in Cell cultures, proliferationand cytokine assay. The factor-specific CD4 T cell response in theLL-FVIII/FIX+LL-mIL-10 group is significantly reduced in comparison tothe control and LL-FVIII/IX groups.

Example B4 IL-10 is Less Effective than LL-IL10 in Potentiating OralTolerance

To assess whether LL-IL10 is as effective as IL-10, mice are orally fedas described above (experimental setting). The factor-specificproliferative CD4 T-cell response is studied in the splenocytes andlymph nodes. The factor-specific CD4 T cell response in theLL-FVIII/FIX+LL-mIL-10 group is significantly reduced in comparison tothe LL-FVIII/IX+IL-10 group.

Example B5 Antigen-Induced T Regulatory Cells FollowingLL-FVIII/FIX-LL-IL10 Combination Therapy Can Transfer Protection fromInhibitor Formation In Vivo

In order to test for active suppression of antibody formation in micetreated with the oral tolerance protocol, we adoptively transfersplenocytes from the different treated groups as described above (Invivo T regulatory activity assay). Compared with controls andLL-FVIII/IX groups, anti-factor IgG formation is significantly reducedin the LL-FVIII/FIX+LL-mIL-10 group, indicating activation of regulatoryCD4⁺ T cells in our combination oral tolerance protocol.

Example C Induction of Tolerance to an Allergen, Der p 1 Following OralAdministration of L. lactis Secreting Said Allergen in Combination withIn Situ Delivered IL-10 Introduction

Allergic asthma is a chronic inflammatory disorder of the airways. It ischaracterized by reversible airway obstruction, elevated serum levels ofallergen-specific immunoglobulin E, mucus hypersecretion and airwayhyperresponsiveness (AHR) to bronchospasmogenic stimuli. Its symptomsare made worse by exposure to an allergen (e.g., tree, grass and weedpollen, dust and dust mites, mold, animal dander) to which the patienthas been sensitized. Type 2 T-helper (Th2) lymphocytes play a crucialrole in the initiation, progression and persistence of the disease.Current data suggest that Th2 responses to allergens are normallysuppressed by regulatory T cells. Furthermore, suppression by thissubset is decreased in allergic individuals. Here, we demonstrate thatoral delivery of allergen in combination with IL-10 producing L. lactissuppresses asthma-like responses via the induction of antigen-specificCD4+ regulatory T cells.

Material and Methods to the Examples

Two Mouse models of allergic asthma that mimics human disease are theOva allergen model and the humanized SCID model.

The Ova Allergen Model

OVA-sensitized mice are inhalationally challenged with OVA aerosol thatleads to Th2 cytokine-dependent eosinophilic airway inflammation,bronchial hyperreactivity, and IgE production, findings highlycharacteristic of human allergic asthma (Brusselle, 1994, Clin ExpAllergy 24:73; Kips et al. 1996, Am J Respir Crit. Care Med 153:535;Brusselle et al. 1995, Am Respir Cell Mol Biol 12:254).

Bacteria

The L. lactis strain MG1363 is used throughout this study. Bacteria arecultured in GM17 medium, i.e. M17 (Difco Laboratories, Detroit, Mich.)supplemented with 0.5% glucose. Stock suspensions of all strains arestored at −20° C. in 50% glycerol in GM17. For intragastricinoculations, stock suspensions are diluted 200-fold in fresh GM17 andincubated at 30° C. They reached a saturation density of 2×10⁹colony-forming units (CFU) per mL within 16 hours. Bacteria areharvested by centrifugation and concentrated 10-fold in BM9 medium. Fortreatment, each mouse receives 100 μL of this suspension daily byintragastric catheter.

Plasmids

The mRNA sequence encoding Gallus gallus Ovalbumin is retrieved fromGenbank (accession number AY223553). Total RNA is isolated from chickenuterus and cDNA is synthesized using 2 μg total RNA, 2 μM oligo dTprimers (Promega Corporation Benelux, Leiden, The Netherlands), 0.01 mMDTT (Sigma-Aldrich, Zwijndrecht, The Netherlands), 0.5 mM dNTP(Invitrogen, Merelbeke, Belgium), 20 U Rnasin (Promega IncorporationBenelux) and 100 U superscript 11 reverse transcriptase (Invitrogen) ina volume of 25 μl. OVA cDNA fragment is amplified by Polymerase ChainReaction (PCR) using the following conditions: 94° C. for 2 min followedby 30 cycles at 94° C. for 45 seconds, 62° C. for 30 seconds and 72° C.for 90 seconds, with the following forward and reverse primers

5′-GGCTCCATCGGTGCAGCAAGCATGGAATT-3′ and5′-ACTAGTTAAGGGGAAAC-ACATCTGCCAAAGAAGAGAA-3′.

The amplified fragment is fused to the Usp45 secretion signal of theerythromycin resistant pT1NX vector, downstream of the lactococcal P1promoter.

MG1363 strains transformed with plasmids carrying murine IL-10 and OVAcDNA are designated LL-IL10 and LL-OVA. LL-pT1NX, which is MG1363containing the empty vector pT1 NX, serve as control.

Quantification of OVA

OVA from LL-OVA are determined using an in house developed OVA-specificenzyme-linked immunosorbent assay (ELISA). Production of the recombinantproteins is also assessed by Western blot analysis.

The OVA Allergen Model Mice

BALB/c mice (6 to 8 weeks of age) are purchased from Charles RiverLaboratories (Calco, Italy). The mice are maintained under specificpathogen-free conditions.

Immunization of Mice

Mice are immunized i.p. with 2 μg of OVA (grade V; Sigma-Aldrich) in 2mg of aluminum hydroxide (alum). This immunization is repeated after a10-day interval (on days 0 and 10). Control mice receive a salineinjection instead of the OVA/alum solution. Seven days after theimmunization, sensitized mice inhale an aerosolized solution of 3% OVAdissolved in PBS for 10 min. OVA inhalation is conducted for 3 days in arow (days 18, 19, and 20). Control mice inhale PBS alone under the sameconditions as used for the experimental group.

Induction of Oral Tolerance

Mice receive LL-OVA alone or combined with IL-10 (1 or 10 μg) orLL-IL10, LL-IL10 alone, IL-10 alone (1 or 10 μg), LL-pT1NX or water (nonfed control). In a prophylactic setting, mice are fed during 2 differentregimes before the first i.p. immunization. Feeding regime 1 and 2consist of 4 and 6 cycles of daily administration for 5 days,alternating with a 2-days period of non-administrating, respectively. Aspositive controls for oral tolerance induction, mice are fed 1 mg(low-dose) or 30 mg (high-dose) of OVA every other day from 10 to 2 daysbefore the first immunization (five feedings in total) by intragastriccatheter that reduce bronchial eosinophilia and airwayhyperresponsiveness, with high dose feeding being more effective thanlow-dose feeding.

In a therapeutic setting, mice are fed daily with the same L. lactisstrains as described for the prophylactic setting, only starting fromthe first immunization to 8 days after the immunization.

As positive control for oral tolerance induction mice are fed 30 mg OVA.

Measurement of Airway Hyperresponsiveness (AHR)

24 h after the final inhalation (day 21), airway hyperresponsiveness isassessed by methacholine-induced airflow obstruction. The mice areexposed for 2.5 min to nebulized physiologic saline (OtsukaPharmaceutical), followed by incremental doses (1-30 mg/ml) of nebulizedmethacholine. These mice are placed in a whole-body plethysmograph for2.5 min following nebulization, and enhanced pause (Penh) is measuredusing Biosystem XA WBP system (Buxco Electronics). “Penh” representspulmonary airflow obstruction and is calculated using the formula:Penh=((Te−Tr)/(Tr×PEF/PIF)), where Penh=enhanced pause (dimensionless),Te=expiratory time (seconds), Tr=relaxation time (seconds), PEF=peakexpiratory flow (milliliters per second), and PIF=peak inspiratory flow(milliliters per second). Penh is measured and averaged approximatelyevery 5 s, and the cumulative values are averaged as the Penh value foreach time point. Airway hyperresponsiveness is expressed as PC200Mch(200% provocative concentration of methacholine), which is theconcentration of methacholine that doubled the baseline Penh value.

Analysis of Bronchoalveolar Lavage Fluid (BALF)

After the measurement of airway hyperresponsiveness, bronchoalveolarlavage samples are obtained. The mice are anesthetized by i.p. injectionof 100 mg/kg ketamin and 10 mg/kg xylazin, and then the lungs arelavaged with 0.5 ml of saline four times. The lavage fluid iscentrifuged and the cells are resuspended in 1 ml of saline with 1% BSA.Total cell numbers are counted using a hemocytometer. Cytospin samplesare prepared by centrifuging the suspensions at 300 rpm for 5 min. Toclearly distinguish the eosinophils from the neutrophils, threedifferent stains are applied: Diff-Quick, May-Grünwald-Giemsa, andHansel (eosin) stains. At least 300 leukocytes are differentiated bylight microscopy based on the standard morphologic criteria. The levelof IL-13, IL-4 and IL-5 in BALF is detected by Cytometric Bead Assay (BDBiosciences, Mountain View, Calif., USA) following the manufacturer'sinstructions.

Measurement of Serum Total IgE and OVA-Specific Ig

On day 21, blood samples are obtained from retro-orbital sinus underanesthesia. After the samples had fully coagulated, they arecentrifuged, and the sera is collected and stored at −80° C. until use.Total IgE is assayed by ELISA using paired Abs (BD Pharmingen) accordingto the manufacturer's instructions. To measure OVA-specific IgE, IgG1,and IgG2a in sera, microtiter plates (Maxisorp, Nunc, VWR International,Haasrode, Belgium) are coated with 2 μg/ml OVA. Subsequently, the wellsare blocked with 0.1% casein in PBS, after which the plates areincubated with mouse serum samples diluted 1:10 to 1:20480 in PBScontaining 0.1% casein and 0.05% Tween 20 (PBS-CT), with goat anti-mouseIgG2a-HRP [Southern Biotechnology Associates (SBA), Imtec ITKDiagnostics, Antwerpen, Belgium, dilution 1:5000], goat anti-mouseIgG1-HRP or goat anti-mouse IgE-HRP (SBA, dilution 1:5000). Afterwashing, substrate [3,3′,5,5′ tetramethylbenzidine (TMB) substratereagent, Pharmingen, Becton Dickinson, Erembodegem, Belgium] is added toeach well. Finally, reactions are stopped by adding 1M H₂SO₄ to thewells. The absorbances are read at 450 nm. ELISA scores are expressed astiters, which are the inverse of the highest dilution that still had onOD₄₅₀ higher than the calculated cutoff value. The cutoff is calculatedas the mean OD₄₅₀ of 5 non-immunized mice increased with three times theSD.

Histological Examination of Lung Tissue

After bronchoalveolar lavage samples are obtained, the lungs areperfused with physiologic saline and are resected from the mice. Thelungs are fixed with neutralized buffered formalin and embedded inparaffin. Sections (3-μm thick) are stained with H&E or periodicacid-Schiff (PAS). The intensity of histological changes in the lungs isevaluated with four grading scores (0, no inflammation; 1, slight/mild;2, moderate; and 3, severe), according to the distribution and intensityof the following findings: 1) epithelial shedding or undulation of thenuclei of bronchial epithelial cells, 2) increase in the number ofgoblet cells, 3) infiltration of inflammatory cells from vessels intothe mucosal and submucosal area of the bronchus and peribronchialinterstitium, and 4) hypertrophy and thickening of the smooth-musclecell layer.

RT-PCR for Analysis of Cytokine and Chemokine Gene Expression in theLung

The lungs are removed after perfusion with physiologic saline, and totalRNA is extracted using ISOGEN (Nippon Gene) according to themanufacturer's instructions. Total RNA (10 μg) is reverse-transcribedusing oligo(dT)15 primer (Promega) and Superscript II RNase H-reversetranscriptase (Invitrogen Life Technologies) at 42° C. for 2 h. Toensure that each sample contained the same amount of cDNA, the MactincDNA concentration of each sample is first determined usingβ-actin-specific primers. These samples are amplified for theappropriate number of cycles, such that the amount of PCR productremained on the linear part of the amplification curve. The PCR productsare electrophoresed in a 2% agarose gel and were visualized by ethidiumbromide staining. The levels of IL-13, eotaxin, IL-10, IFN-γ, and TGF-βare determined using the following specific primer sets.

The sense primer for β-actin 5′-ACGACATGGAGAAGATCTGG-3′, and theantisense primer 5′-TCGTAGATGGGCACAGTGTG-3′. The sense primer for IL-135′-TCTTGCTTGCCTTGGTGGTCTCGC-3′, and the antisense5′-GATGGCATTGCAATTGGAGATGTTG-3′. The sense primer for eotaxin5′-GGGCAGTAACTTCCATCTGTCTCC-3′, and the antisense primer5′-CACTTCTTCTTGGGGTCAGC-3′. The sense primer for IL-105′-TACCTGGTAGGAGTGATGCC-3′, and the antisense5′-GCATAGAAGCATACATGATG-3′. The sense primer for IFN-γ5′-CATAGATGTGGAAGAAAAGA-3′, and the antisense5′-TTGCTGAAGAAGGTAGTAAT-3′. The sense primer for TGF-β5′-CTTTAGGAAGGACCTGGGTT-3′, and the antisense5′-CAGGAGCGCACAATCATGTT-3′.

Cell Cultures, Proliferation and Cytokine Assay

One day after the final inhalation (day 21) single cell suspensions ofspleen and mediastinal lymph nodes are prepared by passing the cellsthrough 70 μm filter cell strainers (Becton/Dickinson Labware).Erythrocytes are removed from the spleen cell suspensions by incubationwith red cell lysis buffer. CD4⁺ T cells and CD4⁺ CD25⁻ T cells areenriched using CD4⁺ T cell isolation kit (Miltenyi Biotec, Germany) orCD4⁺ CD25⁺ regulatory T cell isolation kit (Miltenyi Biotec, Germany),respectively and MACS columns (midiMACS; Miltenyi Biotec).

Proliferation assays of bulk splenocyte and LN populations, 2×10⁵ cellsare cultured in 96-well U-bottom plates in a total volume of 200 μlcomplete medium either alone or with purified OVA, and either with orwithout anti-IL-10 or anti-TGF-β neutralising monoclonal antibodies. OVAis added at concentrations ranging from 1 to 100 μg/ml. The neutralizingantibodies are added at 1, 0.1 and 0.01 μg/ml. For proliferation assaysof CD4⁺ T cells and CD4⁺ CD25⁻ T cell populations, 2×10⁵ cells CD4⁺ Tcells or CD4⁺ CD25⁻ T cells are cultured in 96-well U-bottom plates withmitomycin treated splenocytes that are loaded with 1 mg/ml OVA for 16 h,acting as antigen presenting cells, at ratio's CD4⁺ T cell or CD4⁺ CD25⁻T cell/APCs 1/1, 1/0.3, 1/0.1, 1/0.03, 1/0 in a total volume of 200 μlcomplete medium either with or without neutralizing antibodies. After 72h at 37° C. in a 5% CO₂ humidified incubator, proliferation is assessedby addition of 1 μCi/well [³H]-thymidin. DNA-bound radioactivity isharvested 18 h later onto glass fiber filter mats (Perkin Elmer, Boston,USA) and thymidine-incorporation is measured on a scintillation counter(Perkin Elmer).

For cytokine measurements, supernatants of the cell cultures used in thedifferent proliferation assays is collected after 24, 48 and 72 h ofculture and frozen at −80° C. until cytokine analysis is performed.Cytokine production is quantified using the Mouse InflammationCytometric Bead Assay (BD Biosciences, Mountain View, Calif., USA).

In Vivo T Regulatory Activity Assay

One day after the final inhalation (day 21), spleens of the treated miceare digested with 0.1% collagenase (Sigma-Aldrich) at 37° C. for 20 min.In some experiments, single-cell suspensions of whole spleen cells areprepared and cultured with Con A (2 μg/ml; Sigma-Aldrich) for 48 h.Cells are collected, and 10⁷ cells are adoptively transferred i.v. intonaïve BALB/c mice. For negative selection, CD4⁺, CD8⁺, CD11c⁺, CD19⁺, orCD11b⁺ cells are depleted from the whole spleen cells using magneticbeads (MACS; Miltenyi Biotec) with biotinylated anti-mouse CD4, CD8,CD11c, CD19, and CD11b mAb (BD Pharmingen), according to themanufacturer's instructions. The efficiency of depletion is examined byflow cytometry (>99%). CD4⁺, CD4⁺CD25⁻ cells are purified using CD4⁺ Tcell isolation kit. Regulatory T cell isolation kit following themanufacturer's instructions. The purity of positively selected cells ischecked using flow cytometry. For cell transfer experiments, cells aretransferred into BALB/c mice from the tail veins just before their firstimmunization or just after their second immunization with OVA/alum. Thenumber of transferred cells is 10⁷ for whole spleen cells,subpopulation-depleted spleen cells, or positively selected CD4⁺ cellsand CD4⁺CD25⁻ cells.

In the humanized SCID (hu-SCID) model (as described by Duez et al.,2000; Hammad et al., 2000)

In this model, the allergic immune response to the house dust mite (HDM)allergen Der p 1 can be studied. Such hu-SCID mice reconstituted i.p.with PBMC from HDM-allergic patients and subsequently exposed toaerosols of HDM produce human IgE, develop a pulmonary infiltratecomposed of activated T cells and DCs, and exhibit AHR in response tobronchoconstrictor agents (Pestel et al. 1994, J Immunol, 153:3804; Duezet al., Am J Respir Crit. Care Med. vol 161, ppp 200-206, 2000).

Bacteria

The L. lactis strain MG1363 is used throughout this study. Bacteria arecultured in GM17 medium, i.e. M17 (Difco Laboratories, Detroit, Mich.)supplemented with 0.5% glucose. Stock suspensions of all strains arestored at −20° C. in 50% glycerol in GM17. For intragastricinoculations, stock suspensions are diluted 200-fold in fresh GM17 andincubated at 30° C. They reached a saturation density of 2×10⁹colony-forming units (CFU) per mL within 16 hours. Bacteria areharvested by centrifugation and concentrated 10-fold in BM9 medium. Fortreatment, each mouse receives 100 μL of this suspension daily byintragastric catheter.

Plasmids

Der p 1, a 222 amino-acid residue globular glycoprotein, is one of themajor allergens from Dermatophagoides pteronyssinus (Dpt) mites. DNAsequence with optimal L. lactis codon usage encoding the Der p 1 proteinis synthesized, amplified and fused to the Usp45 secretion signal of theerythromycin resistant pT1NX vector downstream of the lactococcal P1promoter. MG1363 strains transformed with plasmids carrying murineIL-10, Der p 1, Der p 1 aa52-71 and Der p 1 aa117-133 cDNA aredesignated LL-IL10, LL-Derp1, LL-Derp1aa52-71 and LL-Derp1aa117-133.LL-pT1NX, which is MG1363 containing the empty vector pT1NX, serve ascontrol.

Quantification of Der p 1

Der p 1 from LL-Derp1 is determined using an in house developed Der p1-specific enzyme-linked immunosorbent assay (ELISA). Production of therecombinant proteins is also assessed by Western blot analysis.

Patients

Blood is collected from donors sensitive or not sensitive to house dustmites. Allergic patients present the usual features of house dust mitesensitization. Skin prick tests toward Dermatophagoides pteronyssinus(Dpt) allergen (Stallergenes, Fresnes, France)(diameter≧10 mm) arepositive, and all patients have serum specific IgE antibodies. Total IgEconcentrations are greater than 150 IU/ml (150-1600 IU/ml). Healthydonors are tested as negative controls (total IgE levels are less than150 IU/ml, and they have negative skin prick tests toward commonlyinhaled allergens).

Human Peripheral Blood Mononuclear Cell Preparation

Platelet rich plasma is obtained after centrifugation (120×g, 15minutes) and discarded. Blood cells are then diluted in RPMI 1640 (LifeTechnologies, Paisley, Scotland) (vol/vol) and layered over a Ficollgradient (Pharmacia, Uppsala, Sweden). After centrifugation (400×g, 30minutes), PBMCs are harvested at the interface and washed three times insterile RPMI medium before transfer.

Mice

C.B.-17 SCID mice (6-8 weeks old) are maintained in isolators withsterilized bedding in a specific animal facility. The SCID colony isregularly checked for absence of mouse serum immunoglobulins by ELISA.

Peripheral Blood Mononuclear Cells Transfer in SCID Mice: PBMC hu-SCIDMice

SCID mice are between 6 and 8 weeks old at the time of cell transfer.The mice are reconstituted by intraperitoneal injection of 10×10⁶mononuclear cells from allergic patients or healthy donors in 400 μl ofRPMI via a 23-gauge needle. On the same day, they receiveintraperitoneally 2 index reactivity [IR] units Dpt. Four days after thecell reconstitution, SCID mice are exposed to daily allergen aerosolscontaining 100 IR units of Dpt (100 IR units are equivalent toapproximately 200 μg of protein contained in the Dpt extract) for 4successive days (day 0 to day 4). The control group is not exposed toDpt. One day before airway responsiveness measurement (day 35 and day60), hu-SCID mice are exposed to another aerosol of 100 IR units of Dptsolution.

Experimental Setting

Mice receive L. lactis engineered to express Der p 1 or an irrelevantantigen (OVA) as negative control, combined or not with LL-IL10 or IL-10protein (1 or 10 μg).

The engineered L. lactis bacteria are administered orally to SCID miceusing a gastric catheter, using different treatment intervals and dosesstarting one day after PBMC reconstitution. Induction of oral toleranceis assessed by measuring human serum IgE antibodies, analysis ofpulmonary infiltration, measurement of AHR and analysis of cellpopulations and cytokine production in the BALF. Furthermore, inductionof tolerance is assessed by analysis of the proliferative T cellresponse against Der p 1.

Assessment of Airway Responsiveness (AHR)

Airway responsiveness (expressed as provocative dose of carbacholcausing a 50% increase in lung resistance) is measured on day 35 or day60 as described by Duez et al. 2000.

Human IgE Measurements

Several days after transplantation with human cells, mice are bled fromthe retro-orbital sinus under ether anesthesia. Total human IgE isinvestigated by a two-site immuno-radiometric method with the use of twodifferent mouse mAbs specific for the ε-chain (Immunotech International,Luminy, France). At least 20 μl of serum is used in a duplicate test.The sensitivity of the method permits the detection of 0.1 lUiml (0.24ng/ml).

Specific IgE Ab against Dpt allergen is quantified by ELISA. Briefly,plastic tubes (Maxisorb Startube, Nunc, Denmark) are coated overnightwith Dpt allergen in 0.1 M carbonate/bicarbonate buffer (pH 9.6) at 4°C. and saturated with 1% BSA in 0.1 M PBS (pH 7.4) for 2 h at roomtemperature. After washing, the tubes are incubated for 2 h at roomtemperature and overnight at 4° C. with Hu-SCID mice serum diluted inPBS containing BSA (1%) and Tween (0.01%). After extensive washings, aHRP-labeled anti-human IgE Ab is added. After washing, substrate[3,3′,5,5′ tetramethylbenzidine (TMB) substrate reagent, Pharmingen,Becton Dickinson, Erembodegem, Belgium] is added to each well. Finally,reactions are stopped by adding 1 M H₂SO₄ to the wells. The absorbancesare read at 450 nm.

Histological Examination of the Lung.

Lungs are excised at day 35 and fixed in paraformaldehyde and processedfro paraffin embedding. Paraffin tissue sections are stained for thedetection of human CD45+ cells after which human cells on the murinelung sections were quantified by histological scoring as described byDuez et al. 2000.

Analysis of Bronchoalveolar Lavage Fluid (BALF)

BALF is analysed as described in the OVA allergen model.

Cell Cultures, Proliferation and Cytokine Assay:

Single cell suspensions of spleen are prepared by passing the cellsthrough 70 μm filter cell strainers (Becton/Dickinson Labware).Erythrocytes are removed from the spleen cell suspensions by incubationwith red cell lysis buffer. CD4⁺ T cells and CD4⁺ CD25⁻ T cells areenriched using human CD4⁺ T cell isolation kit (Miltenyi Biotec,Germany) or human CD4⁺ CD25⁺ Regulatory T cell isolation kit (MiltenyiBiotec, Germany), respectively and MACS columns (midiMACS; MiltenyiBiotec).

Proliferation assays of bulk splenocyte, 2×10⁵ cells are cultured in96-well U-bottom plates in a total volume of 200 μl complete mediumeither alone or with purified Der p 1, and either with or withoutanti-IL-10 or anti-TGF-β neutralising monoclonal antibodies. Der p 1 isadded at concentrations ranging from 1 to 100 μg/ml. The neutralizingantibodies are added at 1, 0.1 and 0.01 μg/ml. For proliferation assaysof human CD4⁺ T cells and human CD4⁺ CD25⁻ T cell populations, 2×10⁵cells CD4⁺ T cells or CD4⁺ CD25⁻ T cells are cultured in 96-wellU-bottom plates with mitomycin treated human PBMC that are loaded with 1mg/ml Der p 1 for 16 h, acting as antigen presenting cells, at ratio'sCD4⁺ T cell or CD4⁺ CD25⁻ T cell/APCs 1/1, 1/0.3, 1/0.1, 1/0.03, 1/0 ina total volume of 200 μl complete medium either with or withoutneutralizing antibodies. After 72 h at 37° C. in a 5% CO₂ humidifiedincubator, proliferation is assessed by addition of 1 μCi/well[³H]-thymidin. DNA-bound radioactivity is harvested 18 h later ontoglass fiber filter mats (Perkin Elmer, Boston, USA) andthymidine-incorporation is measured on a scintillation counter (PerkinElmer).

For cytokine measurements, supernatants of the cell cultures used in thedifferent proliferation assays is collected after 24, 48 and 72 h ofculture and frozen at −80° C. until cytokine analysis is performed.Cytokine production is quantified using the Human InflammationCytometric Bead Assay (BD Biosciences, Mountain View, Calif., USA).

Example C1 LL-IL10 Significantly Enhances the Tolerance-InducingCapacity of LL-OVA and LL-Der p 1 in OVA- and huSCID Mice Model forAsthma, Respectively

To study the induction of oral tolerance, mice are orally fed asdescribed above (experimental setting). Addition of LL-IL-10significantly enhances the tolerance induction towards OVA/Derp1 as theallergen-specific proliferative response of the splenocytes issignificantly reduced in the LL-OVA/Derp1+LL-mIL-10 group in comparisonto the control and LL-OVA/Derp1 groups.

Example C2 LL-IL10 Potentiates Oral Tolerance in Association withReduced AHR, Eosinophilic Infiltration, Serum IgE Levels, and LoweredIL-13, IL-4 and IL-5 Cytokine Production in Response to Said Allergen

To study the induction of oral tolerance, mice are orally fed asdescribed above (experimental setting). AHR, eosinophilic BALFinfiltration, IgE titer as well as cytokine production in response tosaid factor is determined as described above. AHR, eosinophilic BALFinfiltration, IgE titer is strongly reduced, and IL-13, IL-4 and IL-5significantly lowered in the LL-OVA/Derp1+LL-mIL-10 group in comparisonto the control and LL-OVA/Derp1 groups.

Example C3 LL-IL10 Enhances Oral Tolerance Via CD4⁺ T Cells

To assess whether CD4 T cells mediate the induction of oral tolerance,the allergen-specific proliferative CD4 T-cell response is studied inthe splenocytes and lymph nodes. Therefore, mice are orally fed asdescribed above (experimental setting) and the allergen-specific CD4⁺ Tcell proliferation is determined as described in Cell cultures,proliferation and cytokine assay. The allergen-specific CD4 T cellresponse in the LL-OVA/Derp1+LL-mIL-10 group is significantly reduced incomparison to the control and LL-OVA/Derp1 groups.

Example C4 IL-10 is Less Effective than LL-IL10 in Potentiating OralTolerance

To assess whether LL-IL10 is as effective as IL-10, mice are orally fedas described above (experimental setting). The allergen-specificproliferative CD4 T-cell response is studied in the splenocytes andlymph nodes. The allergen-specific CD4 T cell response in theLL-OVA/Derp1+LL-mIL-10 group is significantly reduced in comparison tothe LL-OVA/Derp1+IL-10 group.

Example C5 Antigen-Induced T Regulatory Cells Following LL-OVA-LL-IL10Combination Therapy Can Transfer Protection from Asthma-Like ResponsesIn Vivo

In order to test for active suppression of asthma-like responses in micetreated with the oral tolerance protocol, we adoptively transfersplenocytes from the different treated groups as described above (Invivo T regulatory activity assay). Compared with controls and LL-OVAgroups, asthma-like responses are significantly reduced in theLL-OVA+LL-mIL-10 group, indicating activation of regulatory CD4⁺ T cellsin our combination oral tolerance protocol.

Example D Induction of Tolerance to Alpha-Gliadin Following OralAdministration of L. lactis Secreting Said Allergen in Combination withIn Situ Delivered IL-10 Introduction

Celiac disease, also known as celiac sprue or gluten-sensitiveenteropathy, is a chronic inflammatory disease that develops from animmune response to specific dietary grains that contain gluten. Celiacis a complex multigenic disorder that is strongly associated with thegenes that encode the human leukocyte antigen variants HLA-DQ2 orHLA-DQ8. One of the most important aspects in the pathogenesis of Celiacis the activation of a T-helper 1 immune response. This arises whenantigen-presenting cells that express HLA-DQ2/DQ8 molecules present thetoxic gluten peptides to CD4(+) T-cells. Both classes of glutenproteins, gliadins and glutenins, contain peptides that bind DQ2 andDQ8. It is generally accepted that the immune response, such as theproduction of IFN-γ from gluten-specific T cells, triggers destructionof the mucosa in the small intestine of celiac disease patients. Hence,the activation of a detrimental immune T cell response in the intestineof celiac disease patients appears to be key in the initiation andprogression of the disease.

Here, we demonstrate that oral delivery of gliadin peptides incombination with IL-10 producing L. lactis suppresses gliadin-specificimmune responses via the induction of antigen-specific CD4⁺ regulatory Tcells.

Material and Methods to the Examples Bacteria

The L. lactis strain MG1363 is used throughout this study. Bacteria arecultured in GM17 medium, i.e. M17 (Difco Laboratories, Detroit, Mich.)supplemented with 0.5% glucose. Stock suspensions of all strains arestored at −20° C. in 50% glycerol in GM17. For intragastricinoculations, stock suspensions are diluted 200-fold in fresh GM17 andincubated at 30° C. They reached a saturation density of 2×10⁹colony-forming units (CFU) per mL within 16 hours. Bacteria areharvested by centrifugation and concentrated 10-fold in BM9 medium. Fortreatment, each mouse receives 100 μL of this suspension daily byintragastric catheter.

Plasmids

DNA sequence with optimal L. lactis codon usage encoding alpha-gliadinprotein (based on sequence of Triticum aestivum, AJ133612), HLA-DQ8(corresponding to the residues 203-220, sequence QYPSGQGSFQPSQQNPQA ofUniProtKB/TrEMBL entry Q9M4L6) and HLA-DQ8 deamidated form(corresponding to the residues 203-220, sequence QYPSGEGSFQPSQENPQA ofUniProtKB/TrEMBL entry Q9M4L6) gliadin peptides are synthesized,amplified and fused to the Usp45 secretion signal of the erythromycinresistant pT1NX vector, downstream of the lactococcal P1 promotor.MG1363 strains transformed with plasmids carrying murine IL-10,alpha-gliadin, HLA-DQ8, and HLA-DQ8 deamidated are designated LL-IL10,LL-HLA/DQ8, LL-HLA/DQ8d. LL-pT1NX, which is MG1363 containing the emptyvector pT1NX, serve as control.

Quantification of HLA-DQ8 and DQ8d

HLA-DQ8 and HLA-DQ8d from LL-HLA/DQ8 and LL-HLA/DQ8d is determined usingan in house developed ELISA. Production of the recombinant proteins isalso assessed by Western blot analysis.

Mice

HLA-DQ8 transgenic mice (Senger et al. 2003) are maintained underspecific pathogen-free conditions on a gluten-free diet and used at theage of 8-14 weeks. Mice are immunized by intrafoodpad injections with 50μg crude gluten (Sigma-Aldrich) in 50 μl CFA (Difco; BD).

Induction of Oral Tolerance

For tolerization experiments, LL-HLA/DQ8, LL-HLA/DQ8d alone or combinedwith IL-10 (1 or 10 μg) or LL-IL10, LL-IL10 alone, IL-10 alone (1 or 10μg), LL-pT1 NX or water (non fed control) is administered before andafter immunisation using different treatment intervals and doses. Aspositive controls for oral tolerance induction, mice are fed 50 mg dosesof wheat gliadin or recombinant alpha-gliadin, dissolved in water fromthe stock solution, on days −7, −6, −5, −4 before immunisation (day 0).

Measurement of Serum Gliadin-Specific Ig

Crude gliadin (Sigma-Aldrich) is resuspended in methanol at 10 mg/ml,and then diluted into absolute ethanol at a concentration of 1 μg/ml.One hundred microliters of the 1 μg/ml gliadin ethanol solution isplaced into each well of an Immulon 2 plate (Fisher ScientificInternational Inc.) and is then allowed to dry under a hood. The plateis then blocked with 4% BSA/PBS for 2 hours at 37° C. The plate iswashed with 1×PBS, 0.05% Tween-20. Sample sera is diluted into 0.1%BSA/PBS 1:200, 1:400, and 1:800 and incubated for 1 hour at 37° C.Detection antibodies are biotinylated rat anti-mouse IgA from AccurateChemical & Scientific Corp., and biotinylated anti-mouse IgG fromJackson ImmunoResearch Laboratories Inc. The enzyme conjugate isstreptavidin-HRP, and the substrate is TMB.

Cell Cultures, Proliferation and Cytokine Assay

Single cell suspensions of spleen and mediastinal lymph nodes areprepared by passing the cells through 70 μm filter cell strainers(Becton/Dickinson Labware). Erythrocytes are removed from the spleencell suspensions by incubation with red cell lysis buffer. CD4⁺ T cellsand CD4⁺ CD25⁻ T cells are enriched using CD4⁺ T cell isolation kit(Miltenyi Biotec, Germany) or CD4⁺ CD25⁺ regulatory T cell isolation kit(Miltenyi Biotec, Germany), respectively and MACS columns (midiMACS;Miltenyi Biotec).

Proliferation assays of bulk splenocyte and LN populations, 2×10⁵ cellsare cultured in 96-well U-bottom plates in a total volume of 200 μlcomplete medium either alone or with crude gliadin or syntheticHLA-DQ8/DQ8d, and either with or without anti-IL-10 or anti-TGF-βneutralising monoclonal antibodies. Antigens are added at concentrationsranging from 1 to 100 μg/ml. The neutralizing antibodies are added at 1,0.1 and 0.01 μg/ml. For proliferation assays of CD4⁺ T cells and CD4⁺CD25⁻ T cell populations, 2×10⁵ cells CD4⁺ T cells or CD4⁺ CD25⁻ T cellsare cultured in 96-well U-bottom plates with mitomycin treatedsplenocytes that are loaded with 1 mg/ml crude gliadin or syntheticHLA-DQ8/DQ8d for 16 h, acting as antigen presenting cells, at ratio'sCD4⁺ T cell or CD4⁺ CD25⁻ T cell/APCs 1/1, 1/0.3, 1/0.1, 1/0.03, 1/0 ina total volume of 200 μl complete medium either with or withoutneutralizing antibodies. After 72 h at 37° C. in a 5% CO₂ humidifiedincubator, proliferation is assessed by addition of 1 μCi/well[³H]-thymidin. DNA-bound radioactivity is harvested 18 h later ontoglass fiber filter mats (Perkin Elmer, Boston, USA) andthymidine-incorporation is measured on a scintillation counter (PerkinElmer).

For cytokine measurements, supernatants of the cell cultures used in thedifferent proliferation assays is collected after 24, 48 and 72 h ofculture and frozen at −80° C. until cytokine analysis is performed.Cytokine production is quantified using the Mouse InflammationCytometric Bead Assay (BD Biosciences, Mountain View, Calif., USA).

Example D1 LL-IL10 Significantly Enhances the Tolerance-InducingCapacity of LL-HLA/DQ8d

To study the induction of oral tolerance, mice are orally fed asdescribed above (Induction of oral tolerance). Addition of LL-IL-10significantly enhances the tolerance induction towards HLA-DQ8d as theHLA-DQ8d-specific proliferative response of the splenocytes issignificantly reduced in the LL-HLA/DQ8d+LL-mIL-10 group in comparisonto the control and LL-HLA/DQ8d groups.

Example D2 LL-IL10 Potentiates Oral Tolerance in Association withReduced Production of IFN-γ in Response to Said Allergen

To study the induction of oral tolerance, mice are orally fed asdescribed above (Induction of oral tolerance). Cytokine production inresponse to HLA-DQ8d is quantified as described above (Cell cultures,proliferation and cytokine assay). In splenocytes and lymph nodes, theproduction of the proinflammatory cytokine IFN-γ is strongly reduced inthe LL-HLA/DQ8d+LL-mIL-10 group in comparison to the control andLL-HLA/DQ8d groups.

Example D3 LL-IL10 Enhances Oral Tolerance Via CD4⁺ T Cells

To assess whether CD4 T cells mediate the induction of oral tolerance,the DQ8-specific proliferative CD4 T-cell response is studied in thesplenocytes and lymph nodes. Therefore, mice are orally fed as describedabove (Induction of oral tolerance) and the DQ8-specific CD4⁺ T cellproliferation is determined as described in Cell cultures, proliferationand cytokine assay. The DQ8-specific CD4 T cell response in theLL-HLA/DQ8d+LL-mIL-10 group is significantly reduced in comparison tothe control and LL-HLA/DQ8d groups.

Example D4 IL-10 is Less Effective than LL-IL10 in Potentiating OralTolerance

To assess whether LL-IL10 is as effective as IL-10, mice are orally fedas described above (Induction of oral tolerance). The DQ8-specificproliferative CD4 T-cell response is studied in the splenocytes andlymph nodes. The DQ8-specific CD4 T cell response in theLL-HLA/DQ8d+LL-mIL-10 group is significantly reduced in comparison tothe LL-HLA/DQ8d+IL-10 group.

Example E Induction of Tolerance to BLG Food Allergen Following OralAdministration of L. lactis Secreting Said Allergen in Combination withIn Situ Delivered IL-10 Introduction

Food allergy is a disease affecting approximately 2% to 5% of thepopulation. In human beings, elevated IgE antibodies as well as thepresence of IL-4-producing, antigen-specific T lymphocytes suggest aTh2-skewed mechanism.

Here, we demonstrate that oral delivery of a food allergen incombination with IL-10 producing L. lactis suppresses allergen-specificimmune responses via the induction of antigen-specific CD4⁺ regulatory Tcells.

Material and Methods to the Examples Bacteria and Plasmids

The L. lactis strain MG1363 is used throughout this study. Bacteria arecultured in GM17 medium, i.e. M17 (Difco Laboratories, Detroit, Mich.)supplemented with 0.5% glucose. Stock suspensions of all strains arestored at −20° C. in 50% glycerol in GM17. For intragastricinoculations, stock suspensions are diluted 200-fold in fresh GM17 andincubated at 30° C. They reach a saturation density of 2×10⁹colony-forming units (CFU) per mL within 16 hours. Bacteria areharvested by centrifugation and concentrated 10-fold in BM9 medium. Fortreatment, each mouse receives 100 μL of this suspension daily byintragastric catheter. Bovine β-lactoglobulin cDNA is amplified andfused to the Usp45 secretion signal of the erythromycin resistant pT1NXvector, downstream of the lactococcal P1 promotor. MG1363 strainstransformed with plasmids carrying murine IL-10 or BLG, are designatedLL-IL10 and LL-BLG. LL-pT1NX, which is MG1363 containing the emptyvector pT1NX, serve as control.

Quantification of Bovine β-Lactoglobulin (BLG)

BLG from LL-BLG are determined using an in house developed BLG-specificenzyme-linked immunosorbent assay (ELISA) and Western blot analysis.

Experimental Setting

The murine model of food allergy used to explore the protective effectof L. lactis is a mouse model of food-induced IgE-type response asdescribed by Frossard et al. (J Allergy Clin Immunol 113:958-964, 2004).Mice receive LL-BLG or an irrelevant antigen (OVA) as negative control,combined or not with LL-IL10 or recombinant IL-10 (1 or 10 μg). As apositive control for tolerance induction, mice receive a high dose ofBLG in the drinking water that prevents the mice from anaphylaxis uponoral challenge with BLG.

In a prophylactic setting, the engineered L. lactis bacteria thatproduce BLG are administered orally to the mice using a gastriccatheter, using different treatment intervals and doses. Subsequently,these recipient mice are orally challenged with purified BLG antigen, inthe presence of cholera toxin. Control animals are exposed to L. lactisengineered with a control vector that does not express BLG (but OVAinstead). Induction of tolerance is assessed by analysis of anaphylaxisafter intragastric antigen challenge, by measuring BLG-specific IgG1,IgG2a and IgE titers in serum and faeces, by determining the number ofantibody secreting cells in spleen and PP, by analysis of the T cellproliferation and cytokine production in MLN, PP and spleen.

To evaluate whether the induction of immune tolerance towards BLG couldbe enhanced by IL-10, mice are administered with LL-BLG along withLL-IL10.

Oral sensitization to BLG.

Four- to 5-week-old female C3H/HeOuJ mice (Charles River) are immunizedat days 0, 7, 14, and 21 by intragastric gavage with 20 mg of BLG(Sigma) and 10 μg of CTX, purchased from List Biological Laboratories in0.2 mol/L NaHCO₃. The positive control group (tolerized mice) receive0.8 mg/mL BLG in their drinking water ad libitum for 4 weeks. The totalamount of protein given (22.4 mg) is similar to the total amount of BLGgiven to the sensitized mice. To demonstrate that the tolerizationprocedure also enduringly activate the peripheral and not only themucosal immune system, a group of tolerized mice is injected twice with80 μg ip BLG adsorbed to 1 mg alum at days 28 and 42.

Antigen Challenge

On day 28, all mice are challenged by intragastric gavage with 100 mgBLG in 0.4 mL 0.2 mol NaHCO₃. Anaphylaxis is observed and graded byusing a reaction score (0, no reaction, to 3, severe reaction or death)described in detail elsewhere (Frossard et al., 2001). The core bodytemperature is measured by infrared at the ear before challenge and 30minutes after gavage. The animals are killed, and blood is collected bycardiac puncture into EDTA-containing tubes, and plasma is obtained forhistamine measurement by commercial ELISA kit (Immunotech, Marseille,France).

Cell Cultures, Proliferation and Cytokine Assay

Single cell suspensions of spleen, mesenteric lymph nodes and PP areprepared as described by Frossard et al. (2004). CD4⁺ T cells and CD4⁺CD25⁻ T cells are enriched using CD4⁺ T cell isolation kit (MiltenyiBiotec, Germany) or CD4⁺ CD25⁺ Regulatory T cell isolation kit (MiltenyiBiotec, Germany), respectively and MACS columns (midiMACS; MiltenyiBiotec). Proliferation assays of bulk splenocyte and LN populations,2×10⁵ cells are cultured in 96-well U-bottom plates in a total volume of200 μl complete medium either alone or with purified BLG, and eitherwith or without anti-IL-10 or anti-TGF-β neutralising monoclonalantibodies. BLG is added at concentrations ranging from 1 to 100 μg/ml.The neutralizing antibodies are added at 1, 0.1 and 0.01 μg/ml. Forproliferation assays of CD4⁺ T cells and CD4⁺ CD25⁻ T cell populations,2×10⁵ cells CD4⁺ T cells or CD4⁺ CD25⁻ T cells are cultured in 96-wellU-bottom plates with mitomycin treated splenocytes that are loaded with1 mg/ml BLG for 16 h, acting as antigen presenting cells, at ratio'sCD4⁺ T cell or CD4⁺ CD25⁻ T cell/APCs 1/1, 1/0.3, 1/0.1, 1/0.03, 1/0 ina total volume of 200 μl complete medium either with or withoutneutralizing antibodies. After 72 h at 37° C. in a 5% CO₂ humidifiedincubator, proliferation is assessed by addition of 1 μCi/well[³H]-thymidin. DNA-bound radioactivity is harvested 18 h later ontoglass fiber filter mats (Perkin Elmer, Boston, USA) andthymidine-incorporation is measured on a scintillation counter (PerkinElmer).

For cytokine measurements, supernatants of the cell cultures used in thedifferent proliferation assays is collected after 24, 48 and 72 h ofculture and frozen at −80° C. until cytokine analysis will be performed.Cytokine production is quantified using the Mouse InflammationCytometric Bead Assay (BD Biosciences, Mountain View, Calif., USA).

In Vivo T Regulatory Activity Assay

In order to test for active suppression of antibody formation in mice,splenocytes, bead-purified CD4⁺ T cells, CD4⁺ CD25⁻ or CD4⁺ CD25⁺ Tcells isolated from the different experimental L. Lactis-treated groupsare adoptively transferred to naïve C3H/HeOuJ mice. Untreated mice areused as control. The number of transferred cells is 107 for whole spleencells, subpopulation-depleted spleen cells, or positively selected CD4⁺cells and CD4⁺ CD25⁻ and CD4⁺ CD25⁺ T cells. If Tregs are implicated,subsequent challenge of these mice with BLG antigen should preventinduction of humoral immune responses against BLG and anaphylaxis.

Enzyme-Linked Immunoassays for BLG-Specific Serum and Feces Antibodies.

Sera are obtained from tail bleedings at day 0, 7, 14, 21 and 28. Fecesare obtained at the same times and resuspended in PBS plus 1% FCS (Lifetechnologies) supplemented with pepstatin 1:1000 (Fluka) at 0.1 mg/mL.The samples are mechanically disaggregated and vortexed for 2 minutes,followed by two centrifugations at 4° C. for 20 minutes at 14,000 rpm.

Sera and feces are assayed for BLG-specific IgE, IgG1, IgG2a and/or IgAantibody levels by a method adapted from Adel-Patient et al. (2000, J.Immunol. Methods). In brief, MaxiSorp microtiter plates (Nunc) arecoated for 18 hours at room temperature with 250 ng/well streptavidin(Fluka), followed by 300 μL of a solution of polyvinylpyroliddon K25(Fluka) overnight. One microgram of biotinylated BLG is incubated for 3hours, and diluted sera (1:6666 and 1:2222 for IgG1, 1:666 and 1:222 forIgG2a, 1:66 and 1:22 for IgE) or feces (1:3, 1:10, and 1:33) in PBS plus10% horse serum is added in duplicates in presence of 0.5 μg/mL goatanti-mouse IgA, rat anti-mouse IgG1 or anti-mouse IgG2aperoxidase-labeled antibodies (Southern Biotechnologies) for 2 hours.For IgE measurement, a monoclonal rat anti-mouse IgE Ab (clone R35-72,BD Pharmingen) followed by peroxidase-coupled anti-rat Ab (Caltag) isadded. Optical density is measured at 490 nm. Results are expressed asarbitrary units, with pooled sera from BLG plus alum-immunized mice usedas a reference serum.

Antigen-Specific Antibody Production Measured by Means of ELISPOT.

Peyer's patches are excised mechanically from the gut and incubated for30 minutes in HBSS medium supplemented with 5 mmol EDTA (LifeTechnologies). Similarly, Peyer patches and mesenteric lymph nodes aregently crushed and filtered through a 70-μm nylon filter. Spleen cellsare preincubated for 5 minutes in Tris-buffered NH₄Cl to remove redblood cells. Lymphoblasts are isolate on a Percoll 60%/66% gradient(Amersham).

For the measurement of BLG-specific IgG1, IgG2a and IgA antibodies,ELISPOT plates (Millipore) are coated with streptavidin overnight at 37°C., followed by addition of 1 μg of biotinylated BLG for 3 hours.Lymphoblasts isolated on a Percoll 60%/66% gradient from are resuspendedat two different concentrations, 1 and 2×10⁶ in Iscove's modifiedDulbecco's medium supplemented with penicillin, streptomycin,L-glutamine, gentamicin, polymixin B. and 5% FCS for 24 hours at 37° C.,followed by overnight incubation at 4° C. with anti-IgA, anti-IgG1 andanti-IgG2a antibodies (Southern Biotechnology). Amino-ethyl-carbazole,100 μL/well, is added for 10 minutes, and the spots are automaticallycounted by using the KS ELISPOT 4.2.1 Software (Zeiss) and expressed ascell-forming units per 10⁶ cells (CFU).

Example E1 LL-IL10 Significantly Enhances the Tolerance-InducingCapacity of LL-BLG in Murine Model of Food Allergy

To study the induction of oral tolerance, mice are orally fed asdescribed above (experimental setting). Addition of LL-IL-10significantly enhances the tolerance induction towards BLG as theallergen-specific proliferative response of the splenocytes issignificantly reduced in the LL-BLG+LL-mIL-10 group in comparison to thecontrol and LL-BLG groups.

Example E2 LL-IL10 Potentiates Oral Tolerance in Association withReduced BLG-Specific Antibody Response and Lowered IL-4 CytokineProduction in Response to Said Allergen

To study the induction of oral tolerance, mice are orally fed asdescribed above (experimental setting). BLG-specific antibody responseand cytokine production in response to said factor is determined asdescribed above. BLG-specific antibodies levels and IL-4 aresignificantly lowered in the LL-BLG+LL-mIL-10 group in comparison to thecontrol and LL-BLG groups.

Example E3 LL-IL10 Enhances Oral Tolerance Via CD4⁺ T Cells

To assess whether CD4 T cells mediate the induction of oral tolerance,the allergen-specific proliferative CD4 T-cell response is studied inthe splenocytes and lymph nodes. Therefore, mice are orally fed asdescribed above (experimental setting) and the allergen-specific CD4⁺ Tcell proliferation is determined as described in Cell cultures,proliferation and cytokine assay. The allergen-specific CD4 T cellresponse in the LL-BLG+LL-mIL-10 group is significantly reduced incomparison to the control and LL-BLG groups.

Example E4 IL-10 is Less Effective than LL-IL10 in Potentiating OralTolerance

To assess whether LL-IL10 is as effective as IL-10, mice are orally fedas described above (experimental setting). The allergen-specificproliferative CD4 T-cell response is studied in the splenocytes andlymph nodes. The allergen-specific CD4 T cell response in theLL-BLG+LL-mIL-10 group is significantly reduced in comparison to theLL-BLG+IL-10 group.

Example E5 Antigen-Induced T Regulatory Cells Following LL-BLG-LL-IL10Combination Therapy Can Transfer Protection from Allergic-Like ResponsesIn Vivo

In order to test for active suppression of allergic-like responses inmice treated with the oral tolerance protocol, we adoptively transfersplenocytes from the different treated groups as described above (Invivo T regulatory activity assay). Compared with controls and LL-BLGgroups, allergic-like responses are significantly reduced in theLL-BLG+LL-mIL-10 group, indicating activation of regulatory CD4⁺ T cellsin our combination oral tolerance protocol.

Example F Induction of Tolerance to Insulin Following OralAdministration of L. lactis Secreting Said Allergen in Combination withIn Situ Delivered IL-10 Introduction

Autoimmunity is characterized by spontaneous inflammatory tissue damageand by impaired physiological function resulting from loss of toleranceto self-antigen. It is associated with a partially overactive immunesystem, which is characterized by an excess of T helper (Th) cells.Predisposing factors, such as susceptibility genes and environmentalfactors are difficult to influence, therefore recent efforts to developimmunotherapies are focused on re-establishing the functional balancebetween pathogenic effector cells and immunoregulatory T cells bydepleting the former and/or enhancing the latter. Autoimmune destructionof pancreatic islet beta cells is the major cause of Type 1 diabetesmellitus (T1D). This destruction is associated with cellular and humoralimmune responses to several beta cell autoantigens, both of which canprecede the clinical onset of disease.

Here, we demonstrate that oral delivery of an autoantigen in combinationwith IL-10 producing L. lactis suppresses diabetic-specific immuneresponses via the induction of antigen-specific CD4⁺ regulatory T cells.

Material and Methods to the Examples Bacteria and Plasmids

The L. lactis strain MG1363 is used throughout this study. Bacteria arecultured in GM17 medium, i.e. M17 (Difco Laboratories, Detroit, Mich.)supplemented with 0.5% glucose. Stock suspensions of all strains arestored at −20° C. in 50% glycerol in GM17. For intragastricinoculations, stock suspensions are diluted 200-fold in fresh GM17 andincubated at 30° C. They reach a saturation density of 2×10⁹colony-forming units (CFU) per mL within 16 hours. Bacteria areharvested by centrifugation and concentrated 10-fold in BM9 medium. Fortreatment, each mouse receives 100 μL of this suspension daily byintragastric catheter. DNA sequence with optimal L. lactis codon usageencoding the human proinsulin II B24-C36 peptide (hpIIp), porcineinsulin and immunodominant-peptide InsB₉₋₂₃ (B9-23 is essentially thesame across many species human, rat and mouse) are synthesized,amplified and fused to the Usp45 secretion signal of the erythromycinresistant pT1 NX vector, downstream of the lactococcal P1 promotor.

MG1363 strains transformed with plasmids carrying murine IL-10, hpIIp,Insulin, InsB₉₋₂₃ are designated LL-IL10, LL-hpIIp, LL-insulin,LL-InsB₉₋₂₃. LL-pT1NX, which is MG1363 containing the empty vectorpT1NX, served as control. Expression of these proteins is determinedusing antigen-specific ELISA and Western blot analysis.

Mice

Non-obese female and male diabetic (NOD) mice and NOD-severe combinedimmunodeficient (SCID) (Balb/c background) mice are purchased from theJackson laboratory. Balb/c wild type (WT) mice are purchased fromCharles River Italy. Mice are maintained in a specific pathogen-freecentral animal facility. Mice are treated and used in agreement with theinstitutional guidelines.

Experimental Setting

In a prophylactic setting, the LL-hpIIp, LL-insulin, LL-InsB₉₋₂₃ areadministered orally to NOD mice starting from day 21 of age (weaning),alone or along with LL-IL10 or recombinant mouse IL-10 (1-10 μg), andusing the optimal feeding regime or until 100 days of age (when mostmice develop diabetes). In addition, LL-pT1NX are administered orally asa negative control. For the positive (tolerizing) control group,3-week-old NOD mice are treated orally with 0.8 mg human insulin for 3times a week for 2 or 4 weeks. Development of diabetes is determined bycontinuous monitoring of urine glucose levels three times a week and incase of glucosuria monitoring of blood glucose levels. Pancreases arecollected at 12-23 weeks and at the end of experiment (35 weeks), andserial sections are stained with hematoxylin/eosin to score mononuclearcell infiltration or by immunohistochemistry to analyse T cellinfiltration. In a therapeutic setting the LL-hpIIp, LL-insulin,LL-InsB₉₋₂₃ are administered orally, alone or along with LL-IL10 orrecombinant mouse IL-10, to diabetic NOD females showing stableglycosuria and hyperglycemia (12-23 weeks). In addition, LL-pT1NX isadministered orally as a negative control. For the positive (tolerizing)control group, diabetic NOD mice are treated as described in Bresson etal. 2006. Complete remission is defined as the disappearance ofglycosuria and a return to normal glycemia.

The precise mechanisms of tolerance induction are analyzed in vitro, invivo after re-challenging the NOD mice with specific autoantigens and byadoptive T-cell transfer into NOD-SCID mice.

Detection of Diabetes:

Glucose monitoring: urine glucose is measured by using Diastix (Miles)and is confirmed by blood glucose measurements with the blood glucosemonitoring system OneTouch Ultra (LifeScan Inc.). Diabetes is defined as2 consecutive blood glucose values superior to 250 mg/dl.

Insulitis: Mice are killed by CO₂ asphyxiation and the pancreas is fixedin 10% formalin overnight, embedded in paraffin, and serial 5 μmsections are stained with haematoxylin and eosin. The insulitis score(mean±SD) is determined by microscopically grading the degree ofcellular infiltration in 10-15 islets/mouse as follows: 0, no visiblesign of islet infiltration; 1, peri-islet infiltration; 2, <50%infiltration; 3, >50% infiltration.

Immunohistochemistry

To detect insulin, CD4 and CD8 expression in pancreatic 1 cells, primaryAbs (guinea pig anti-swine insulin from Dako [dilution 1:300], anti-CD4RM4.5 and anti-CD8a IHC from BD Biosciences [dilution 1:50] are appliedto frozen tissue sections as described in Christen et al., 2004.

In Vitro Proliferation Assay

Single cell suspensions of spleen, mesenteric LN (MLNs) and PLNs areprepared. Proliferation assays of total splenocyte populations, 2×10⁵cells are cultured in 96-well U-bottom plates in a total volume of 200μl complete medium either alone or with graded concentrations (1-100μg/ml) of purified human insulin or peptides specific for CD4 T cells(InsB₉₋₂₃, H-2^(d or g) restricted) or for CD8 T cells (InsB₁₅₋₂₃, K^(d)restricted) (Sigma), and either with or without anti-IL-10 or anti-TGF-βneutralising monoclonal antibodies. The neutralizing antibodies areadded at 1, 0.1 and 0.01 μg/ml. For proliferation assays of total CD3⁺ Tcells, CD8⁺ T cells, CD4⁺ T cells and CD4⁺ CD25⁻ T cell populations,0.2×10⁵ cells T cells are cultured in 96-well U-bottom plates with 1×10⁵irradiated splenocytes from WT Balb/c mice loaded with insulin or GAD65or peptides specific for CD4⁺ or CD8⁺ T cells, in a total volume of 200μl complete medium either with or without neutralizing antibodies. After72 hr at 37° C. in a 5% CO₂ humidified incubator, proliferation isassessed by addition of 1 μCi/well [³H]-thymidin. DNA-boundradioactivity is harvested 16-18 hr later onto glass fiber filter mats(Perkin Elmer, Boston, USA) and thymidine-incorporation is measured on ascintillation counter (Perkin Elmer). T-cells are purified from PLNs orspleens by negative selection through magnetic bead separation usingCD3⁺, CD4⁺ or CD8⁺ isolation kit (MACS; Milteny Biotec, Auburn, Calif.).CD4⁺ T cells are used as total cells or further separated into CD25⁺ andCD25⁻ by MACS using CD25⁺ isolation kit (Milteny Biotec). The purity(>90%) of the cell populations is determined by flow cytometricanalysis.

For cytokine measurements, supernatants of the cell cultures used in thedifferent proliferation assays (antigen-specific stimulation), describedabove, are collected after 72 h of culture and frozen at −80° C. untilcytokine analysis is performed. Cytokine production is quantified usingthe Mouse Inflammation Cytometric Bead Assay (BD Biosciences, MountainView, Calif., USA). Purified CD3⁺ T cells, CD4⁺ T or CD8⁺ T cells arecultured and stimulated in vitro non-specifically with ananti-CD3/anti-CD28 mixture (1 μg/ml each) for 24 hours or they remainedunstimulated as control. The supernatants is harvested, and analysed forIL-10, IL-4, IL-5 and IFNγ production using BD™ Cytometric Bead Arrayflex set on a BD FACSArray Bioanalyzer using the FCAP array software (BDBiosciences). Capture ELISA experiments are used to determine TGF-β1using the Quantikine kit (R&D Systems).

In Vitro T Cell Proliferation Inhibition Assay

2×10⁴ purified total splenic CD4⁺ CD25⁻ T cells isolated from recentlydiabetic female NOD (8-12 weeks) are co-cultured with varying numbers ofCD8⁺ T cells, CD4⁺ T cells and CD4⁺ CD25⁻ T cell populations isolatedfrom the spleen, MLN or PLNs from the different experimental groups inthe presence of 2×10⁴ T-cell depleted irradiated insuline- orpetides-loaded splenocytes from WT Balb/c mice. After 72 hr at 37° C. ina 5% CO₂ humidified incubator, proliferation is assessed by addition of1 μCi/well [³H]-thymidin. DNA-bound radioactivity is harvested 16-18 hrlater onto glass fiber filter mats (Perkin Elmer, Boston, USA) andthymidine-incorporation measured on a scintillation counter (PerkinElmer).

In Vitro Cytotoxicity Assay

Lymphoblast targets used are Con A-activated splenocytes from BALB/cmice. A total of 106 target cells are labelled with 100 μCi of ⁵¹Cr(Amersham International, Buckinghamshire, U.K) for 90 min at 37° C.,washed three times and then incubated with 1 μg/ml peptide (InsB₁₅₂₃ oran irrelevant peptide) at 37° C. for 1 h. Target cells are washed twotimes and seeded at 10⁴ cells per well. CD8⁺ T cells, isolated fromspleen, MLNs and PLNs are added to each well, in triplicate, at variouseffector:target (E:T) ratios. The plates are centrifuged at 500 rpm for2 min, and incubated at 37° C. for 4 h. After incubation, supernatantsare collected for determination of ⁵¹Cr release [% lysis=100×(testcpm−spontaneous cpm)/(total cpm−spontaneous cpm)]. For the indirectkilling assay, CD8⁺ T cells are incubated with 5 μg/ml anti-CD3 antibody(clone 145-2C11, Pharmingen) prior to incubation with effectors.

Adoptive Transfer of Diabetes

NOD-SCID mice at 8-10 wk are injected i.v. with 2×10⁷ or i.p. with 5×10⁶splenocytes isolated from diabetic female NOD mice (6 weeks, 12 weeksand 18 weeks) combined with or without graded numbers of bead-purifiedCD3⁺ T cells, CD8⁺ T cells, CD4⁺ T cells, CD4⁺ CD25⁻ or CD4⁺ CD25⁺ Tcells isolated from the different experimental L. Lactis-treated groups.Untreated mice are used as control. Development of diabetes isdetermined by continuous monitoring of blood glucose levels three timesa week.

Example F1 LL-IL10 Significantly Enhances the Tolerance-InducingCapacity of LL-hpIIp, LL-Insulin, LL-InsB₉₋₂₃ in the Non-Obese DiabeticMouse

To study the induction of oral tolerance, mice are orally fed asdescribed above (experimental setting). Addition of LL-IL10significantly enhances the tolerance induction towards autoantigen asthe autoantigen-specific proliferative response of the splenocytes issignificantly reduced in the LL-hpIIp/insulin/InsB₉₋₂₃+LL-mIL-10 groupin comparison to the control and LL-hpIIp/insulin/InsB₉₋₂₃ groups.

Example F2 LL-IL10 Potentiates Oral Tolerance in Association withReduced Insulitis, Deceased Rate of Beta Cell Destruction, and IncreasedIL-10 Production by Splenocytes

To study the induction of oral tolerance, mice are orally fed asdescribed above (experimental setting). The presence of insulitis, therate of beta-cell destruction and cytokine production in response tosaid autoantigen is determined as described above. Histological analysisshows a significant lower degree of insulitis and beta cell destructionand increased IL-10 production in theLL-hpIIp/insulin/InsB₉₋₂₃+LL-mIL-10 group in comparison to the controland LL-hpIIp/insulin/InsB₉₋₂₃ groups.

Example F3 LL-IL10 Enhances Oral Tolerance Via CD4⁺ T Cells

To assess whether CD4 T cells mediate the induction of oral tolerance,the autoantigen-specific proliferative CD4 T-cell response is studied inthe splenocytes and lymph nodes. Therefore, mice are orally fed asdescribed above (experimental setting) and the autoantigen-specific CD4⁺T cell proliferation is determined as described (in vitro proliferationassay). The autoantigen-specific CD4 T cell response in theLL-hpIIp/insulin/InsB₉₋₂₃+LL-mIL-10 group in comparison to the controland LL-hpIIp/insulin/InsB₉₋₂₃ groups.

Example F4 IL-10 is Less Effective than LL-IL10 in Potentiating OralTolerance

To assess whether LL-IL10 is as effective as IL-10, mice are orally fedas described above (experimental setting). The autoantigen-specificproliferative CD4 T-cell response is studied in the splenocytes andlymph nodes. The autoantigen-specific CD4 T cell response in theLL-hpIIp/insulin/InsB₉₋₂₃+LL-mIL-10 group in comparison to theLL-hpIIp/insulin/InsB₉₋₂₃+IL-10 group.

Example F5 Autoaggressive CD8⁺ Responses are Suppressed in NOD MiceFollowing LL-InsB₁₅₋₂₃-LL-IL10 Combination Therapy

To examine whether our combination approach induce suppressive CD4⁺ Tcells that are capable of modulating diabetes by bystander suppressivemechanisms, we analyze the effect on CD8⁺ autoaggressive T cells. Thepercentage and/or activity of antigen-specific autoaggressive CD8⁺ cellsis strongly reduced after combination therapy.

Example F6 Antigen-Induced T Regulatory Cells FollowingLL-InsB₁₅₋₂₃-LL-IL10 Combination Therapy can Transfer Protection fromAllergic-Like Responses In Vivo

In order to test for active suppression of diabetic-like responses inmice treated with the oral tolerance protocol, we adoptively transfersplenocytes from the different treated groups as described above(adoptive transfer of diabetes). Compared with controls and LL-InsB₉₋₂₃group, diabetic-like responses are significantly reduced in theLL-InsB₉₋₂₃+LL-mIL-10 group, indicating activation of regulatory CD4⁺ Tcells in our combination oral tolerance protocol.

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1. A method to induce immune tolerance or to treat an immune responserelated disease comprising delivering a composition comprising anantigen and an immuno-modulating compound expressing microorganism to amammal in need thereof by mucosal delivery whereby immune tolerance isinduced or an immune response related disease is treated.
 2. (canceled)3. The method according to claim 1, wherein said mammal is selected fromthe group consisting of mouse, rat, dog, cat, cattle, horse, pig andhuman.
 4. The method according to claim 1, wherein said immune toleranceis at least 1.5 times more than immune tolerance before said induction.5. The method according to claim 1, wherein said induction of immunetolerance is measured by modulation of a cytokine level in said mammal.6. The method according to claim 5, wherein said modulation is anincrease of a cytokine level, wherein said cytokine is selected from thegroup consisting of IL-10, TGF-β, IL-4 and IFNα.
 7. The method accordingto claim 6, wherein said increase of the cytokine level is at least 1.5times more than the cytokine level before said induction.
 8. The methodaccording to claim 5, wherein said modulation is a decrease of acytokine level, wherein said cytokine is selected from the groupconsisting of IFN-γ, IL-2, IL-5, IL-6, IL-12, IL-13, TNF-α, and MCP-1.9. The method according to claim 8, wherein said decrease of thecytokine level is at least 1.5 times less than the cytokine level beforesaid induction.
 10. The method according to claim 1, wherein saidantigen provokes a T-cell mediated and/or B-cell mediated immuneresponse.
 11. The method according to claim 10, wherein said T-cellmediated immune response is a Th1 immune response or a Th2 immuneresponse.
 12. (canceled)
 13. The method according to claim 1, whereinsaid antigen is an allergen, allo-antigen, self-antigen or auto-antigen.14. The method according to claim 1, wherein said antigen is involved inthe induction of allergic asthma, multiple sclerosis, type I diabetes,autoimmune uveitis, autoimmune thyroiditis, autoimmune myastheniagravis, rheumatoid arthritis, food allergy or celiac disease.
 15. Themethod according to claim 1, wherein said antigen is Anti-CD3.
 16. Themethod according to claim 1, wherein said antigen is delivered by anantigen expressing micro-organism.
 17. The method according to claim 16,wherein said antigen is displayed at the surface of said antigenexpressing micro-organism.
 18. The method according to claim 16, whereinsaid antigen is secreted. 19-20. (canceled)
 21. The method according toclaim 1, wherein said mucosal delivery is selected from the groupconsisting of rectal delivery, buccal delivery, pulmonary delivery,ocular delivery, nasal delivery, vaginal delivery and oral delivery. 22.The method according to claim 1, wherein said antigen and/or saidimmuno-modulating compound expressing micro-organism are delivered byspray, capsule, aerosol, lozenges, bolus, tablet, sachets, liquid,suspension, emulsion or troches.
 23. The method according to claim 1,wherein said antigen and said immuno-modulating compound expressingmicro-organism are delivered in a unit dosage form, selected from atablet, capsule or metered aerosol dose.
 24. The method according toclaim 1, wherein said antigen is delivered simultaneously with orsequential to said immuno-modulating compound expressing microorganism.25. The method according to claim 1, wherein said immuno-modulatingcompound is an immuno-suppressing compound.
 26. The method according toclaim 25, wherein said immuno-suppressing compound is selected from atolerance enhancing cytokine, a tolerance enhancing antibody and animmuno-suppressing cytokine or antibody.
 27. (canceled)
 28. The methodaccording to claim 1, wherein said immuno-modulating compound is animmuno-suppressing cytokine selected from the group consisting of IL-4,IL10, IFN-α, Flt3L, TGFβ and RANK-L.
 29. The method according to claim1, wherein said immuno-modulating compound is an immuno-suppressingantibody selected from the group consisting of anti-IL-2, anti-IL12 andanti-IFN-γ.
 30. The method according to claim 1, wherein saidmicro-organism is a bacterium or yeast.
 31. The method according toclaim 30, wherein said bacterium is a lactic acid bacterium.
 32. Themethod according to claim 31, wherein said lactic acid bacterium isselected from the group consisting of Lactobacillus, Leuconostoc,Pediococcus, Lactococcus, Streptococcus, Aerococcus, Carnobactehum,Enterococcus, Oenococcus, Teragenococcus, Vagococcus, and Weisella. 33.The method according to claim 32, wherein said Lactococcus isLactococcus lactis.
 34. The method according to claim 30, wherein saidyeast is Saccharomyces cerevisiae.
 35. The method according to claim 30,wherein said micro-organism is a probiotic micro-organism.
 36. Themethod according to claim 1, wherein said immuno-modulating compound issecreted from the micro-organism.
 37. The method according to claim 1,wherein said immuno-modulating compound is displayed on the surface ofthe micro-organism.
 38. The method according to claim 1, wherein saidantigen and said immuno-modulating compound are expressed by the samemicro-organism.
 39. The method according to claim 1, wherein saidantigen and/or said immuno-modulating compound expressing micro-organismfurther comprises an adjuvant, pharmaceutical acceptable carrier and/orexcipient.
 40. The method according to claim 1, wherein said antigenand/or said immuno-modulating compound expressing micro-organism furthercomprises a compound stimulating production of immuno-suppressingcytokines.
 41. The method according to claim 40, wherein said compoundstimulating production of immuno-suppressing cytokines is cholera toxinB subunit.
 42. The method according to claim 1, wherein said antigenand/or said immuno-modulating compound secreting micro-organism isdelivered for at least 1 day.
 43. The method according to claim 1,wherein said antigen and said immuno-modulating compound secretingmicro-organism are delivered at least once a day.
 44. The methodaccording to claim 1, wherein said antigen and/or said immuno-modulatingcompound secreting micro-organism is delivered in a dose of 10 femtogramto 100 mg per day.
 45. A composition comprising an immuno-modulatingcompound secreting micro-organism in combination with an antigen. 46.The composition according to claim 45, wherein said composition is apharmaceutical composition.
 47. The composition according to claim 45,wherein said antigen is an allergen, allo-antigen, self-antigen orauto-antigen.
 48. The composition according to claim 45, wherein saidantigen is involved in the induction of allergic asthma, multiplesclerosis, type I diabetes, autoimmune uveitis, autoimmune thyroiditis,autoimmune myasthenia gravis, rheumatoid arthritis, food allergy orceliac disease.
 49. The composition according to claim 45, wherein saidantigen is Anti-CD3.
 50. The composition according to claim 45, whereinsaid antigen is delivered by an antigen expressing micro-organism. 51.The composition according to claim 50, wherein said antigen is displayedat the surface of said antigen expressing micro-organism.
 52. Thecomposition according to claim 45, wherein said antigen is secreted. 53.The composition according to claim 45, wherein said antigen and/or saidimmuno-modulating compound expressing micro-organism are present in aspray, capsule, aerosol, lozenges, bolus, tablet, sachets, liquid,suspension, emulsion or troches.
 54. The composition according to claim45, wherein said antigen and said immuno-modulating compound expressingmicro-organism are present in a unit dosage form, for example a tablet,capsule or metered aerosol dose.
 55. The composition according to claim45, wherein said immuno-modulating compound is an immuno-suppressingcompound.
 56. The composition according to claim 55, wherein saidimmuno-suppressing compound is selected from a tolerance enhancingcytokine, a tolerance enhancing antibody, and an immuno-suppressingcytokine or antibody.
 57. The composition according to claim 45, whereinsaid immuno-modulating compound is a cytokine selected from the groupconsisting of IL-4, IL10, IFN-α, Flt3L, TGFβ and RANK-L.
 58. Thecomposition according to claim 45, wherein said immuno-modulatingcompound is an immuno-suppressing antibody selected from the groupconsisting of anti-IL-2, anti-IL12 and anti-IFN-γ.
 59. The compositionaccording to claim 45, wherein said micro-organism is a probioticmicro-organism.
 60. The composition according to claim 45, wherein saidmicro-organism is a bacterium or yeast.
 61. The composition according toclaim 60, wherein said bacterium is a lactic acid bacterium.
 62. Thecomposition according to claim 61, wherein said lactic acid bacterium isselected from the group consisting of Lactobacillus, Leuconostoc,Pediococcus, Lactococcus, Streptococcus, Aerococcus, Carnobacterium,Enterococcus, Oenococcus, Teragenococcus, Vagococcus, and Weisella. 63.The composition according to claim 62, wherein said Lactococcus isLactococcus lactis.
 64. The composition according to claim 60, whereinsaid yeast is Saccharomyces cerevisiae.
 65. The composition according toclaim 45, wherein said antigen and said immuno-modulating compound areexpressed by the same micro-organism.
 66. The composition according toclaim 45, further comprising an adjuvant, pharmaceutical acceptablecarrier and/or excipient.
 67. The composition according to claim 45,further comprising a compound stimulating production ofimmuno-suppressing cytokines.
 68. The composition according to claim 67,wherein said compound stimulating production of immuno-suppressingcytokines is cholera toxin B subunit.
 69. The composition according toclaim 45, wherein said antigen and/or said immuno-modulating compoundsecreting micro-organism are present in a dose of at least at least 10femtogram to 100 mg. 70-72. (canceled)